Method for isolating poly (A) nucleic acids

Abstract

The present invention relates to a method for isolating a poly (A) nucleic acid. Specifically, the present invention relates to a method of isolating a poly (A) nucleic acid having a single chain poly (A) extension from a sample containing a nucleic acid, comprising: (a) providing a hybridization composition comprising: i) a sample containing a nucleic acid; ii) a hybridization solution comprising: aa. A sodium salt; bb. Quaternary ammonium salt; wherein the components of the hybridization solution can be added into the sample as a single solution or can be respectively added into the sample in any sequence; iii) a capture probe capable of hybridizing to a poly (A) extension of a poly (A) nucleic acid; and incubating the hybridization composition under conditions to form a nucleic acid hybrid between the poly (A) nucleic acid and the capture probe; and (b) separating the formed hybrid from the remaining sample. The methods are particularly useful for the efficient isolation of poly (A) RNA from different samples while avoiding the residue of unwanted non-poly (A) nucleic acids (e.g., rRNA). The invention also provides beneficial methods, hybridization solutions and kits.

Description

[0001] This application is a divisional application of the application with international application date of April 28, 2015, international application number PCT / EP2015 / 059117, which entered the Chinese national phase on October 31, 2016, application number 201580023578.1, and invention name “Method for isolating poly(A) nucleic acid”. Technical Field

[0002] The present invention provides a method for isolating poly(A) nucleic acids from a sample containing nucleic acids. The method effectively enriches poly(A) nucleic acids, such as poly(A) RNA, while removing unwanted non-poly(A) nucleic acids, such as rRNA. The method is particularly suitable for preparing poly(A) RNA for next generation sequencing (NGS) applications. In addition, compositions and kits suitable for performing the methods of the present invention are also provided. Background Art

[0003] The present disclosure relates to the isolation of polyadenylated nucleic acids, in particular poly(A) RNA. Polyadenylation generally refers to the addition of an extension consisting of multiple (usually tens to hundreds) consecutive adenine (A) residues to a biological molecule, wherein the extension is generally present at the 3' end of the molecule, generally referred to as a "poly(A) tail". Nucleic acids containing respective poly(A) extensions or poly(A) tails are generally referred to as "poly(A) nucleic acids". In eukaryotes, polyadenylation is a process associated with the production of mature messenger RNA (mRNA) for gene expression (translation). Here, a poly(A) tail is added to RNA at the end of transcription (nuclear polyadenylation). In eukaryotes, almost all mRNAs have a poly(A) tail at the 3' end, with only a few exceptions, such as animal replication-dependent histone mRNAs (see, e.g., Lopez et al. (RNA 14(1): 1-10, 2007)). In addition, in addition to the vast majority of mRNAs, various eukaryotic non-coding RNAs are also polyadenylated. This also includes some small RNAs, such as microRNAs, which may have their intermediate forms of poly(A) tails during microRNA maturation. In addition, nucleic acids can also be artificially polyadenylated to provide them with poly(A) tails. The poly(A) tails of nucleic acids have been used as a means of separating poly(A) nucleic acids from different sample types, in particular for separating poly(A) nucleic acids from nucleic acids without poly(A) tails, also referred to herein as "non-poly(A) nucleic acids".

[0004] Poly(A) nucleic acids can be isolated with the aid of probes that are capable of hybridizing to single-stranded poly(A) stretches. Such probes can capture poly(A) nucleic acids by hybridizing to the poly(A) stretches, hereinafter also referred to as capture probes. Typically, an oligonucleotide is used for this purpose, the oligonucleotide containing a sequence complementary to the poly(A) stretch of poly(A) nucleic acid, herein also referred to as capture oligonucleotides. In order to simplify the separation process of the captured poly(A) nucleic acid, a solid support functionalized with a capture probe is typically used. The standard approach for the specific isolation of poly(A) RNA from total RNA is based on the method of Aviv and Leder (1972). Here, a short stretch of a complementary DNA oligonucleotide ("oligo dT", "oligo dT") is fixed to an insoluble matrix, which is then used as a selective immobilization matrix for poly(A) RNA by establishing conditions that are favorable for the formation of RNA-DNA duplexes. The total RNA sample was loaded onto a column with an appropriate salt buffer (initially 10 mM Tris, pH 7.5 containing 0.5 M KCl) to allow the capture probe to hybridize with the poly(A) tail. The column was then washed thoroughly with an application buffer (containing 0.5 M KCl), followed by a low ionic strength solution (0.1 M KCl), and then the mRNA was eluted with 10 mM Tris (pH 7.5).

[0005] Subsequent modifications to the original method retain the basic process of hybridization with immobilized oligo-dT, but have changed the format, for example, from a column to a batch procedure, to allow the procedure to be performed more quickly and have used NaCl or LiCl as salt. A further change is to use plastic or glass beads as the solid phase instead of cellulose and to use magnetic beads as the solid support. The quality of this magnetism allows such magnetic beads to be separated in batches by means of a magnet. In addition, biotin-streptavidin connections have been used to establish the connection between oligo-dT and the solid support, wherein the oligonucleotide is biotinylated and the solid support is covalently coupled to streptavidin. Hybridization can be performed in solution, and in a subsequent step the oligo-dT-mRNA hybrid is connected to the solid support. This method tends to be an inefficient method for selectively isolating poly (A) RNA while removing rRNA. The residual level of rRNA is usually high enough to provide the same problems as total RNA, especially for the analysis of rare transcripts.

[0006] Other methods for the separation of poly(A) nucleic acids aim to enhance interactions in extended A:T hybrids by using low ionic strength (high stringency) washes and attempt to find more "inert" materials for use as supports. Isostabilizing agents, such as quaternary ammonium salts belonging to the class of tetramethylammonium (TMA+) and tetraethylammonium (TEA+) ions and betaine, a derivative of the glycine amino acid, balance the hydrogen bonding strength of A:T and G:C base pairs when used at appropriate concentrations (Jacobs et al., 1988; Jacobs et al., 1985; Gitschier et al., 1986; Melchior et al., 1973; Rees et al., 1993; Wood et al., 1985; WoZney, 1990). Such isostabilizing agents are used in the art to facilitate poly(A) separations. US 6,812,341 describes a method for enriching poly(A) with the aim of removing rRNA residues during the separation process by using equivalent stabilizers such as tetramethylammonium (TMA+) and tetraethylammonium (TEA+) ions, preferably TMAC or TEAC. WO 90 / 12116 relates to a general method in which magnetic particles coated with oligo-dT are used for the separation of poly(A) RNA. Here, tetraalkylammonium cations are combined with chaotropic salts to stabilize the A:T bond between the poly(A) tail of the poly(A) nucleic acid and the oligo(dT) probe.

[0007] In addition, various commercial products are available for poly(A) nucleic acid isolation, such as the MagAtract® Direct M48 kit, which uses oligo(dT) capture oligonucleotides immobilized on magnetic beads, or the Oligotex® mRNA kit (Qiagen) for isolation of poly(A) RNA, which uses oligo(dT) capture oligonucleotides immobilized on polystyrene-latex beads as a solid support.

[0008] There is still a great need for efficient poly(A) nucleic acid isolation methods (i.e., efficient capture of poly(A) nucleic acids while removing non-poly(A) nucleic acids). These methods, for example, provide poly(A) RNA requirements suitable for next generation sequencing (NGS) applications performed in a massively parallel manner. NGS technology platforms have in common that they require the preparation of sequencing libraries suitable for massively parallel sequencing. Most platforms follow a general library preparation procedure with only minor modifications before "running" on the instrument. This procedure includes fragmenting DNA, which can be obtained from cDNA, followed by DNA repair and end polishing (blunt ends or A overhangs), and finally, usually the ligation of platform-specific adapters. The preparation and design of such sequencing libraries are described, for example, in Voelkerding et al. (Clinical Chemistry 55: 4 641-658, 2009) and Metzker (Nature Reviews / Genetics Volume 11, January 2010, pages 31-46). NGS has also been used for transcriptome sequencing. The study of the entire transcriptome using next generation sequencing technology provides a detailed, high-throughput view of the transcriptome. Transcriptome sequencing is also known as RNA sequencing (RNA-seq) and is used, for example, to map and quantify transcripts in biological samples. The technology has been rapidly adopted in the study of diseases such as cancer.

[0009] Preparing sequencing libraries from poly(A) RNA has the following advantages: RNA species that do not carry poly(A) tails, such as rRNA (which is not of interest), are not theoretically recovered and therefore are not carried into the sequencing reaction. Therefore, most of the sequences obtained from sequencing libraries generated using poly(A) RNA correspond to protein-encoding mRNAs that carry poly(A) tails. However, in eukaryotic cells, only about 1 to 5% of the total RNA consists of primary transcripts, i.e., polyadenylated mRNAs, while ribosomal RNA (rRNA) constitutes about 90% of this RNA species. Therefore, even when starting from poly(A) RNA, a major problem in transcriptome sequencing is the presence of interfering RNA molecules, especially if the poly(A) RNA starting material does not have sufficient purity and contains non-poly(A) contamination. If a large amount of rRNA is involved in library construction, sequencing functions will be used to sequence these ubiquitous molecules. Highly abundant rRNA may dominate sequencing reads, thereby hindering the study of low-expressed genes and wasting valuable sequencing resources. In addition, the presence of ribosomal RNA may result in low signal-to-noise ratios, which can make it difficult to detect RNA species of interest. Therefore, improvements in the removal of rRNA and / or other unwanted non-poly(A) RNA during the isolation of poly(A) RNA increase the value of downstream sequencing because more information can be derived from the sequencing run.

[0010] However, although there are many methods in the art that can be used to separate poly (A) nucleic acids, the prior art methods all have disadvantages. There is usually a trade-off between poly (A) nucleic acid recovery and achieving the removal of unwanted non-poly (A) nucleic acids. Methods that effectively remove unwanted non-poly (A) nucleic acids often suffer from poor poly (A) nucleic acid recovery rates. Other methods achieve good poly (A) nucleic acid recovery rates, but the removal of non-poly (A) nucleic acids such as rRNA is insufficient. In fact, available methods usually require two or more rounds of poly (A) nucleic acid enrichment to provide poly (A) nucleic acid samples, wherein the amount of non-poly (A) nucleic acids, for example, rRNA is low enough to provide useful samples for certain applications, such as NGS applications with higher purity requirements. This requirement of repeating the entire poly (A) nucleic acid separation procedure can lead to several adverse side effects, and in addition, the number of hours required to perform the procedure is also extended. The representative distribution of various poly (A) nucleic acids can change again and again, or the inevitable loss associated with the repeated procedure can reduce the level of low-abundance information usually sought beyond the detection limit. These shortcomings pose major problems especially in diagnostic settings and NGS applications, where simplicity, efficient non-poly(A) nucleic acid removal, speed and reliability are driving features for viable poly(A) nucleic acid isolation methods, especially poly(A) RNA isolation methods.

[0011] An object of the present invention is to provide an improved method for isolating poly (A) nucleic acids, such as, in particular, poly (A) RNA. Specifically, an object of the present invention is to provide a method for isolating poly (A) nucleic acids, which effectively enriches poly (A) nucleic acids while effectively removing non-poly (A) nucleic acids. In addition, an object is to provide an improved method for isolating poly (A) nucleic acids for next generation sequencing. In addition, products suitable for performing various methods are provided. Summary of the invention

[0012] The present invention is based in particular on the surprising discovery that nucleic acids containing single-stranded poly(A) extensions (hereinafter also referred to as "poly(A) nucleic acids") can be effectively and specifically separated using capture probes and hybridization conditions involving the use of a sodium salt in combination with a quaternary ammonium salt. When the hybridization conditions described herein are used, contamination of non-poly(A) nucleic acids is reduced. As demonstrated in the examples, the hybridization conditions used in the method according to the present invention ensure effective capture and separation of poly(A) nucleic acids, while significantly reducing the binding of unwanted non-poly(A) nucleic acids and the resulting residues in the isolated poly(A) nucleic acids. This results in the recovery of highly selective poly(A) nucleic acids being achieved even in one separation / enrichment step. This also increases the speed of the process compared to prior art methods that require two or more separation cycles. The present invention can be used to specifically and effectively separate poly(A) RNA from a sample containing nucleic acids, while removing unwanted non-poly(A) RNA, such as rRNA, during the separation process. The method is particularly suitable for preparing poly(A) RNA for next generation sequencing (NGS) applications (such as transcriptome sequencing). For such applications, it is particularly important to efficiently capture poly(A) RNA while removing non-poly(A) RNA, because the more RNAs of no interest, such as rRNA, are eliminated from the isolated poly(A) RNA, the more information is obtained from a sequencing run. Therefore, the methods described herein make an important contribution to the art.

[0013] In addition, it has been found that the favorable hybridization conditions using a sodium salt in combination with a quaternary ammonium salt not only provide stringent and selective capture conditions for poly(A) nucleic acids, but also provide favorable washing conditions. Therefore, the hybridization conditions described herein can be advantageously used to remove non-poly(A) nucleic acids that may be bound to the capture probe and / or the captured poly(A) nucleic acid during washing.

[0014] Therefore, according to a first aspect, there is provided a method for isolating a poly(A) nucleic acid having a single-stranded poly(A) stretch from a sample containing nucleic acid, comprising:

[0015] (a) providing a hybridization composition comprising:

[0016] i) samples containing nucleic acids;

[0017] ii) a hybridization solution comprising:

[0018] aa. Sodium salt;

[0019] bb. quaternary ammonium salt;

[0020] wherein the components of the hybridization solution may be added to the sample as a single solution or may be added to the sample separately in any order;

[0021] iii) a capture probe capable of hybridizing to a poly(A) stretch of a poly(A) nucleic acid;

[0022] and incubating the hybridization composition under conditions such that a nucleic acid hybrid is formed between the poly(A) nucleic acid and the capture probe;

[0023] (b) Separating the formed hybrids from the remaining sample.

[0024] According to a second aspect, there is provided a method for sequencing a poly(A) nucleic acid, comprising:

[0025] (a) isolating poly(A) nucleic acid from a sample containing nucleic acid using the method according to the first aspect;

[0026] (b) Sequencing the isolated poly(A) nucleic acid molecules.

[0027] This method is particularly suitable for sequencing poly(A) RNA.

[0028] According to a third aspect, there is provided a water-soluble hybridization solution suitable for hybridizing a poly(A) nucleic acid with a capture probe capable of hybridizing to a poly(A) stretch of the poly(A) nucleic acid, the hybridization solution comprising:

[0029] aa. Sodium salt, concentration ≤ 500 mM;

[0030] bb.Quaternary ammonium salt.

[0031] The hybridization solution of the third aspect can be used in combination with the method of the first, second and fourth aspects of the present invention and used to perform the method thereof. It is particularly suitable for establishing binding conditions for hybridizing poly (A) nucleic acid with capture probes while preventing non-poly (A) nucleic acid hybridization. In addition, it can be used to provide stringent washing conditions to remove bound non-poly (A) nucleic acids during the washing step while maintaining hybridization of poly (A) nucleic acids with capture probes, thereby improving the purity of isolated poly (A) nucleic acids.

[0032] According to a fourth aspect, there is provided a kit for isolating poly(A) nucleic acid from a sample containing nucleic acid, comprising:

[0033] (a) a hybridization solution according to the third aspect;

[0034] (b) a capture probe capable of hybridizing to the poly(A) stretch of the poly(A) nucleic acid.

[0035] According to a fifth aspect, there is provided a method for isolating a poly(A) nucleic acid having a single-stranded poly(A) extension from a sample containing nucleic acid, comprising:

[0036] (a) hybridizing a poly(A) nucleic acid with a capture probe capable of hybridizing to a poly(A) stretch of the poly(A) nucleic acid, thereby forming a nucleic acid hybrid between the poly(A) nucleic acid and the capture probe;

[0037] (b) separating the formed hybrid from the remaining sample;

[0038] (c) washing the separated hybrids with a hybridization solution according to the third aspect, wherein the components of the hybridization solution may be added to the hybrids as a single solution, or may be added separately to the hybrids in any order to produce the hybridization solution for washing;

[0039] (d) releasing the poly(A) nucleic acid from the washed hybrid.

[0040] Here, the beneficial hybridization solution comprising sodium salt and quaternary ammonium salt provided by the present invention is used in the washing step during the separation process to reduce the contamination of non-poly (A) nucleic acid in the washing step. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The ΔCt values ​​of the tested conditions are shown (ΔCt values ​​were calculated as the mean Ct values ​​determined after poly(A) RNA enrichment minus the mean Ct values ​​measured from the initial total RNA samples).

[0042] Figure 2a ) to d) show the ΔCt values ​​obtained with the tested conditions (calculated as described above).

[0043] Figure 3a ) shows the ΔCt values ​​obtained for 18S and 28S rRNA, Figure 3b ) shows the ΔCt values ​​obtained for GAPDH and PPIA target mRNAs, Figure 3c ) shows the ΔΔCt value of GAPDH, and Figure 3d ) shows the ΔΔCt values ​​of PPIA.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention is based, inter alia, on the surprising discovery that poly(A) nucleic acids can be effectively isolated using capture probes that can hybridize to poly(A) stretches (e.g., poly(A) tails) of poly(A) nucleic acids and hybridization conditions involving the use of a sodium salt in combination with a quaternary ammonium salt. The hybridization conditions described herein ensure good poly(A) nucleic acid recovery while reducing the amount of unwanted non-poly(A) nucleic acids in the isolated poly(A) nucleic acids.

[0046] Method for isolating poly(A) nucleic acid

[0047] In a first aspect, a method is provided for isolating a poly(A) nucleic acid having a single-stranded poly(A) stretch from a sample containing nucleic acid, comprising:

[0048] (a) providing a hybridization composition comprising:

[0049] i) samples containing nucleic acids;

[0050] ii) a hybridization solution comprising:

[0051] aa.Sodium salt;

[0052] bb. quaternary ammonium salt;

[0053] wherein the components of the hybridization solution may be added to the sample as a single solution, or may be added to the sample separately in any order;

[0054] iii) a capture probe capable of hybridizing to the poly(A) stretch of the poly(A) nucleic acid; and incubating the hybridization composition under conditions such that a nucleic acid hybrid is formed between the poly(A) nucleic acid and the capture probe;

[0055] (b) Separating the formed hybrids from the remaining sample.

[0056] The main advantages are as described above. The steps and preferred embodiments of each method will be described later.

[0057] Step (a)

[0058] In step (a), a hybridization composition is provided, which comprises i) a sample comprising nucleic acids, wherein the separation of poly(A) nucleic acids is to be performed, ii) a hybridization solution comprising a sodium salt and a quaternary ammonium salt, and iii) a capture probe capable of hybridizing to a poly(A) stretch of the poly(A) nucleic acid. The hybridization composition is incubated under conditions that allow the formation of nucleic acid-hybrids between the poly(A) nucleic acid and the capture probe.

[0059] The nucleic acid-containing sample for separating poly (A) nucleic acid can be any sample that often separates poly (A) nucleic acid such as poly (A) RNA. Details about the sample containing nucleic acid and non-restrictive common examples are also described below. According to one embodiment, poly (A) RNA is separated from total RNA.

[0060] Firstly, the hybridization conditions will be explained subsequently, as they are decisive for achieving efficient binding of the poly(A) nucleic acids to the capture probes, while the non-poly(A) nucleic acids are not able to bind efficiently and are therefore effectively removed during the separation process.

[0061] Hybridization compositions and hybridization solutions

[0062] The composition of the hybridization composition and the hybridization conditions therefrom will be explained subsequently. Providing the hybridization composition comprises adding a hybridization solution to the sample. The components of the hybridization solution are preferably added to the sample as a single solution. However, the components of the hybridization solution may also be added to the sample separately in any order, for example, using two or more solutions, each solution containing at least one reagent, such as a sodium salt and / or a quaternary ammonium salt of the hybridization solution, to produce a "hybridization solution" within the meaning of the present disclosure. This method also provides a "hybridization solution", which is therefore also included in this term.

[0063] Then, suitable and preferred embodiments of hybridization compositions and hybridization solutions suitable for establishing these hybridization compositions are disclosed.

[0064] When describing and defining the concentrations of components of the "hybridization solution" in the hybridization composition of the present invention in the specification and claims of this application, the volume contributed by the capture probe and the solid support (if the added volume of the solid support is used to assist in the capture and separation steps) is not taken into account when determining the concentrations of the components in the hybridization composition described subsequently. That is, when calculating the concentrations of the individual components in the hybridization composition, the capture probe and any solid support or other binding agent used to assist in separation (if used) are ignored. In addition, when describing and defining the concentrations of components of the "hybridization solution" in the hybridization composition of the present invention, according to one embodiment, the corresponding components, such as sodium chloride or quaternary ammonium salts, which are not added via the hybridization solution but are included in the sample containing nucleic acids (if included therein), are not taken into account when calculating the concentrations of the respective components. The concentrations and concentration ranges specified below are calculated ignoring the composition of the nucleic acid-containing sample, as described herein, preferably a purified (or possibly diluted) nucleic acid sample. However, according to another embodiment, the various components of the nucleic acid-containing sample, such as sodium salt and / or quaternary ammonium salt, are taken into account in the calculation. In this embodiment, the salt concentration in the hybridization composition can be adjusted by adding a hybridization solution to obtain the final concentrations of sodium salt and quaternary ammonium salt (provided by the nucleic acid-containing sample and the hybridization solution) in the hybridization composition given in the following description.

[0065] According to one embodiment, the hybridization solution comprises a component in concentrated form, which allows it to be diluted with a sample containing nucleic acid and / or a diluent solution, so that when mixed with a sample nucleic acid and / or a diluent solution, a suitable final concentration is reached in the hybridization composition. Using a concentrated hybridization solution has the following advantages: the user is more flexible relative to the volume of the acid-containing sample material, and the total volume to be processed remains low. For example. The amount of the nucleic acid-containing sample can be adjusted to a certain volume by adding a diluent solution such as water or other suitable solvents, and then the volume is mixed with a hybridization solution of a predetermined volume to provide hybridization conditions of the hybridization composition. The method has advantages, because only by adding an appropriate amount of suitable diluent solution to the nucleic acid-containing sample, the same volume of (possibly diluted) sample material can be provided to the hybridization solution of the predetermined volume. For example, 1 volume of (possibly diluted) nucleic acid-containing sample can be contacted with 1-10, 1-5, 1-3 or 1-2 volumes of hybridization solution, depending on the composition of the hybridization solution. A particularly preferred ratio is 1 volume of (possibly diluted) nucleic acid-containing sample and 1 volume of hybridization solution (1:2 dilution), and is also used in several embodiments.

[0066] The hybridization solution and its hybridization composition contain one (a) sodium salt. This also includes using a mixture of different sodium salts as one (a) sodium salt. The sodium salt promotes the binding of poly (A) nucleic acid to the capture probe. It can be an inorganic or organic sodium salt. According to one embodiment, the sodium salt is not a chaotropic salt (chaotropic salt, a salt with a high chaotropic sequence), and therefore, the sodium salt is a non-chaotropic sodium salt. According to this embodiment, the hybridization solution or hybridization composition does not contain any chaotropic sodium salt. According to one embodiment, the sodium salt is a sodium halide. Preferably, the sodium halide is sodium chloride. It has been found that sodium chloride is particularly suitable because it provides favorable hybridization conditions when used according to the teachings of the present invention.

[0067] During hybridization, the sodium salt must be present in a concentration sufficient to promote the hybridization of the poly (A) nucleic acid with the capture probe. Therefore, the hybridization composition comprises a certain concentration of hybridization solution sodium salt, which effectively promotes the hybridization of the poly (A) nucleic acid with the capture probe. However, for non-poly (A) nucleic acid contaminants that need to be reduced, it is found that reducing the concentration of sodium salt in the hybridization composition is advantageous. Therefore, according to one embodiment, the hybridization composition comprises a hybridization solution sodium salt with a concentration of ≤250mM. The hybridization composition may comprise a hybridization solution sodium salt selected from the following concentrations: 25mM-250mM, 35mM-200mM, 40mM-175mM, 50mM-150mM, 55mM-125mM, 60mM-125mM and 60mM-100mM.

[0068] According to one embodiment, the hybridization solution for setting up hybridization conditions in the hybridization composition comprises a sodium salt of concentration ≤ 500mM. The hybridization solution may comprise a sodium salt selected from the following concentration ranges: 50mM-500mM, 75mM-400mM, 85mM-350mM, 100mM-300mM, 115mM-250mM, 120mM-225mM and 125mM-200mM. According to one embodiment, the sodium salt concentration that the hybridization solution comprises is located in the range selected from 125mM-175mM. As demonstrated in the examples, when contacted with an isopyknic sample (or a diluted sample), this hybridization solution provides particularly good results. Therefore, hybridization conditions can be set up in the hybridization composition described in the preceding paragraph. As mentioned above, sodium chloride is preferably used as a sodium salt.

[0069] According to a preferred embodiment, the hybridization solution sodium salt concentration contained in the hybridization composition is ≤200mM, ≤175mM, ≤150mM, ≤125mM or ≤100mM. Such lower concentrations of sodium salt in the hybridization composition are preferred because they provide stringent conditions for specific hybridization of poly(A) nucleic acids to capture probes, while non-specific hybridization of non-poly(A) nucleic acids is greatly reduced. As shown in the examples, the reduction in the sodium salt concentration in the hybridization solution / hybridization composition has a significant effect on the binding of non-poly(A) nucleic acids. Their binding is significantly reduced, which results in less contamination of non-poly(A) in the isolated poly(A) nucleic acids. Relatively speaking, the reduction in the salt concentration has a smaller effect on the hybridization of poly(A) nucleic acids with capture probes. Therefore, relatively speaking, the effect of reducing the concentration of sodium salt on unwanted non-poly(A) nucleic acids is more serious than that on poly(A) nucleic acids. However, reducing the salt concentration also reduces the yield of poly(A) nucleic acids and thus has an adverse effect on them, which is not desirable.

[0070] The inventors were surprised to find that when a quaternary ammonium salt was additionally included in the hybridization solution and its hybridization composition, this adverse effect on the poly (A) nucleic acid yield associated with the reduction in sodium salt concentration in the hybridization composition was overcome. Therefore, this combination maintains the beneficial effect of removing non-poly (A) nucleic acids in the poly (A) nucleic acid separation process, while overcoming and compensating for the reduction in the poly (A) nucleic acid yield associated with the reduction in sodium concentration. Therefore, hybridization conditions are provided using a combination of sodium salts such as sodium chloride and quaternary ammonium salts, which advantageously provide good poly (A) nucleic acid yields while effectively reducing the contamination of non-poly (A) nucleic acids in the separated poly (A) nucleic acids.

[0071] Therefore, the hybridization solution and its hybridization composition contain one (a) quaternary ammonium salt. This also includes using a mixture of different quaternary ammonium salts as one (a) quaternary ammonium salt. According to one embodiment, the quaternary ammonium salt is a tetraalkylammonium salt. The tetraalkylammonium salt can be a tetramethylammonium salt (TMA) or a tetraethylammonium salt (TEA). Suitable tetraalkylammonium salts include, but are not limited to, tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetraethylammonium nitrate (TEAN), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB). According to one embodiment, the quaternary ammonium salt is not tetramethylammonium sulfate.

[0072] According to a preferred embodiment, the quaternary ammonium salt is a tetraalkylammonium salt selected from tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB). Preferably, tetramethylammonium bromide is used as the quaternary ammonium salt.

[0073] During the hybridization process, the quaternary ammonium salt is present in a concentration that can be combined with the sodium salt to support the binding of the poly (A) nucleic acid to the capture probe. According to one embodiment, the hybridization composition contains a hybridization solution quaternary ammonium salt at a concentration of ≤3M, ≤2.5M, ≤2M or ≤1.5M. The quaternary ammonium salt concentration that the hybridization composition may contain is ≥100mM, ≥125mM, ≥250mM or ≥375mM. Preferably, the quaternary ammonium salt concentration contained in the hybridization composition is selected from 0.1M-1.75M, 0.125M-1.5M, 0.25M-1.25M, 0.375M-1M and 0.375M-0.75M. As described above, the quaternary ammonium salt is preferably a tetraalkylammonium salt, and suitable examples are as described above.

[0074] According to one embodiment, the quaternary ammonium salt concentration included in the hybridization solution for setting up hybridization conditions in the hybridization composition is ≤6M, ≤5M, ≤4M or ≤3M. The quaternary ammonium salt concentration that the hybridization solution can include is ≥200mM, ≥250mM, ≥500mM or ≥750mM. Preferably, the quaternary ammonium salt concentration included in the hybridization solution is selected from 0.2M-3.5M, 0.25M-3M, 0.5M-2.5M, 0.75M-2M and 0.75M-1.5M. As shown in the examples, when, for example, an equal volume (or diluted) sample is contacted, this type of hybridization solution provides particularly good results. Therefore, hybridization conditions can be set up in the hybridization composition described in the preceding paragraph.

[0075] As demonstrated in the examples, each concentration of sodium salt (preferably sodium chloride) and quaternary ammonium salt (preferably tetraalkylammonium salt such as tetramethylammonium bromide) in hybridization compositions and / or hybridization solutions provides particularly good results. Suitable concentrations can also be determined by those skilled in the art based on the teachings provided herein.

[0076] The hybridization solution and / or hybridization composition may contain other components, non-limiting embodiments of which are described subsequently.

[0077] According to one embodiment, the hybridization composition comprises a detergent. As demonstrated in the examples, when following the teachings of the present invention, a detergent is not required. However, it can be used, for example, to support the denaturation of the secondary structure in the poly (A) nucleic acid, thereby prompting the capture probe to hybridize with the poly (A) tail. The detergent can be selected from ions, zwitterions and non-ionic detergents. Various detergents and their use in hybridization reactions are well known to those skilled in the art. According to one embodiment, an ionic detergent is used. Ionic detergents include anionic and cationic detergents. As demonstrated in the examples, hybridization compositions comprising anionic detergents such as SDS or LiDS provide good results. Suitable concentration ranges of detergents in the hybridization composition include, for example: 0.025%-5%, and can be selected from 0.05%-3%, 0.75%-2.5%, 0.1%-2.25% and 0.15%-2%. The detergent is preferably included in the hybridization solution and is therefore added along with the hybridization solution. The suitable concentration range of the detergent in the hybridization solution includes, for example, 0.05%-10%, and can be selected from 0.1%-7.5%, 0.25%-5%, 0.5%-3% and 0.75%-2%. In addition, the lower concentration ranges of 0.1%-2%, 0.15%-1% and 0.2%-0.5% are also suitable. However, the detergent can also be derived from the processing of the sample, depending on the sample type processed. For example, if the lysate is processed as a sample containing nucleic acid, and a detergent is used to lyse the sample. However, a detergent is not necessary. Therefore, according to one embodiment, the hybridization composition does not contain a detergent.

[0078] In addition, the hybridization composition may include a chelating agent. Chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylene glycoltetraacetic acid (EGTA) and N, N-bis(carboxymethyl)glycine (NTA), and citrate or oxalate in addition. According to a preferred embodiment, EDTA is used as a chelating agent. As used herein, the term "EDTA" particularly refers to the EDTA portion of an EDTA compound, such as Na2EDTA, K2EDTA or K3EDTA. Suitable concentration ranges of chelating agents in the hybridization composition include, for example, 0-0.25M, 0.5mM-50mM, 0.75mM-10mM and 1mM-5mM. The chelating agent is preferably included in the hybridization solution and thereby added by the hybridization solution. According to one embodiment, the chelating agent concentration included in the hybridization solution is selected from 0-0.5M, 1mM-100mM, 1.5mM-20mM and 2mM-10mM. However, chelators may also originate from sample processing, depending on the type of sample processed.

[0079] According to one embodiment, the hybridization solution comprises a buffer, such as Tris or other biological buffers such as MOPS, HEPES, MES or BIS-TRIS. Examples of suitable buffers are also known to those skilled in the art.

[0080] The pH value of the hybridization solution may be within the range selected from 6 to 9. The hybridization solution may further comprise water or other solvents suitable for dissolving the components of the hybridization solution.

[0081] In addition, as mentioned above, water or another suitable diluent solution can be added to the sample respectively to dilute the sample, and be adjusted to a certain volume for (diluted) nucleic acid-containing sample, then it is mixed with a certain volume of hybridization solution. This dilution has also diluted the hybridization solution, thereby helps to set up hybridization conditions in the hybridization composition. As described herein, the advantage that corresponding dilution has is that the hybridization solution can be provided in concentrated form. In addition, different sample volumes can be easily processed with the hybridization solution of the same amount. In order to provide hybridization composition, specific sample volume can be filled to a specific volume with diluent solution such as water, then the hybridization solution with for example equal amount (or other predetermined) volume will be mixed. It is also possible to adopt the sample / dilution sample of other ratios for hybridization according to the composition of the hybridization solution.

[0082] According to one embodiment, the hybridization solution does not contain chaotropic ions.

[0083] The hybridization conditions used in the method according to the present invention are based on the balanced combination of sodium salt and quaternary ammonium salt, which causes the effective separation of poly (A) nucleic acid, while preventing the residual of non-poly (A) residues. Therefore, except sodium salt and quaternary ammonium salt, hybridization solution and each hybridization composition do not contain other hybridization promoting salts with the concentration that will offset these beneficial effects. Therefore, according to embodiments, hybridization solution and / or hybridization composition do not contain hybridization promoting salts with the concentration that will offset the beneficial effect obtained by the combination of sodium salt and quaternary ammonium salt, such as lithium chloride or potassium chloride or other non-sodium halides, MgCl and / or chaotropic salts. In embodiments, the concentration of the salt (if present) is 100mM or less, 75mM or less, 50mM or less or 25mM or less in the hybridization composition and / or hybridization solution. Preferably, the hybridization solution does not contain any hybridization promoting salt except sodium salt and quaternary ammonium salt.

[0084] Non-limiting preferred embodiments of hybridization compositions and hybridization solutions, particularly with respect to sodium salts (preferably sodium chloride) and quaternary ammonium salts (preferably tetraalkylammonium salts) contained therein, are described below. As described above, when describing the concentration of components of the hybridization solution in the hybridization composition, the volume contributed by the capture probes and the device used to assist in separation, particularly the solid support (if used to assist in capture and separation), is not considered when determining the concentration of the components in the hybridization composition. In addition, as described above, the components of the hybridization solution can be added to the sample as a single solution, or can be added to the sample separately in any order, for example, using two or more solutions containing at least one of the chemicals of the hybridization solution to produce the hybridization solution.

[0085] According to one embodiment, the hybridization composition comprises a sodium salt of the hybridization solution at a concentration of ≤250 mM and a tetraalkylammonium salt as a quaternary ammonium salt. In order to ensure effective binding, the concentration should be ≥25 mM, preferably ≥35 mM, more preferably ≥50 mM. In order to provide the respective hybridization compositions, a hybridization solution can be used, which comprises a sodium salt at a concentration of ≤500 mM and a tetraalkylammonium salt as a quaternary ammonium salt. According to one embodiment, the hybridization solution comprises a sodium salt at a concentration of ≥50 mM, preferably ≥75 mM, more preferably ≥100 mM.

[0086] According to one embodiment, the hybridization composition comprises a sodium salt of the hybridization solution in a concentration selected from 25mM-175mM, 50mM-150mM, 55mM-125mM, 60mM-115mM and 60mM-100mM, and a tetraalkylammonium salt as a quaternary ammonium salt in a concentration selected from 0.25M-1.25M, 0.375M-1M and 0.375M-0.75M salt. In order to provide a corresponding hybridization composition, a hybridization solution can be used, which comprises a sodium salt in a concentration selected from 50mM-350mM, 100mM-300mM, 115mM-250mM, 120mM-225mM and 125mM-200mM, and a tetraalkylammonium salt and as a quaternary ammonium salt in a concentration selected from 0.5M-2.5M, 0.75M-2M and 0.75M-1.5M.

[0087] According to one embodiment, the hybridization composition comprises a sodium salt of the hybridization solution at a concentration selected from 37.5mM-125mM, 50mM-100mM and 55mM-87.5mM and 60mM-100mM, and a tetraalkylammonium salt as a quaternary ammonium salt at a concentration selected from 0.25M-1.25M, 0.375M-1M and 0.375M-0.75M, wherein the sodium salt is sodium chloride. In order to provide a corresponding hybridization composition, a hybridization solution can be used, which comprises sodium chloride in a concentration selected from 75 mM-250 mM, 100 mM-200 mM and 125 mM-175 mM, and a tetraalkylammonium salt selected from the group consisting of tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB) as a quaternary ammonium salt, in a concentration selected from 0.5 M-2.5 M, 0.75 M-2 M, and 0.75 M-1.5 M. As mentioned, the use of tetraethylammonium bromide (TEAB) is particularly preferred.

[0088] As mentioned above, a detergent may be additionally included in the hybridization composition and may be introduced via the hybridization solution. The details are described above and reference is made to the corresponding disclosure.

[0089] Capture probe

[0090] The hybridization composition also comprises one (a) capture probe capable of hybridizing with a single-stranded poly (A) extension of a poly (A) nucleic acid. The capture probe may be at least partially complementary to a single-stranded poly (A) extension of a poly (A) nucleic acid, thereby allowing the poly (A) nucleic acid to hybridize via the poly (A) extension (e.g., especially their poly (A) tail) to capture the probe. Typically, the capture probe comprises a single-stranded sequence that can hybridize with a poly (A) extension of a poly (A) nucleic acid. It may also consist of such a sequence. The hybridization composition comprising "one (a)" capture probe used herein also encompasses an embodiment in which the hybridization composition comprises two or more different capture probes.

[0091] Preferably, the capture probe is a capture oligonucleotide. The capture oligonucleotide sequence is designed to provide sufficient complementarity with the poly(A) stretch of the poly(A) nucleic acid to allow specific hybridization with the poly(A) stretch sufficiently strong for subsequent separation procedures, wherein the captured poly(A) nucleic acid is separated from non-poly(A) nucleic acids and other contaminants. Capture oligonucleotides suitable for this purpose are well known in the art and therefore do not require any detailed description. Even so, some non-limiting examples are described subsequently.

[0092] The capture probe may comprise or consist of RNA, DNA, PNA (peptide nucleic acid), LNA (locked nucleic acid) and / or other analogs. In particular, analogs of the nucleobases T or U may be used as long as they are capable of hybridizing with A residues. Any capture probe capable of hybridizing with a poly(A) extension of a poly(A) nucleic acid (i.e., such as being capable of sequence-specific binding with a poly(A) nucleic acid) is considered a "capture probe". As described above, the capture probe is preferably a capture oligonucleotide, such as a synthetic oligonucleotide. The capture oligonucleotide may comprise a polypyrimidine sequence capable of hybridizing with a poly(A) extension (e.g., a poly(A) tail of a poly(A) nucleic acid or a portion thereof), thereby being capable of capturing a poly(A) nucleic acid by hybridization. According to an embodiment, the capture oligonucleotide comprises at least a continuous unit extension complementary to the poly (A) chain extension, such as a nucleic acid base or an analog thereof, and the extension of the capture oligonucleotide is preferably at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40 units of length known in the prior art. Longer extensions of at least 50 or more units can also be used in the capture oligonucleotide. Preferably, the capture oligonucleotide is at least in the region complementary to the single-stranded poly (A) extension. The entire oligonucleotide can also be single-stranded and / or complementary to the poly (A) extension or the poly (A) tail, respectively. According to one embodiment, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of all pairing units (e.g., bases) of the capture region and / or the entire capture oligonucleotide are capable of hybridizing with at least a portion of the poly(A) stretch of the poly(A) nucleic acid. According to one embodiment, when the capture oligonucleotide hybridizes with the poly(A) stretch of the poly(A) nucleic acid, a double-stranded nucleic acid hybrid is formed that does not contain any mismatches.

[0093] According to one embodiment, the capture oligonucleotide is a poly (T) or poly (U) nucleic acid molecule. Preferably, a poly (dT) nucleic acid molecule is used. The term poly (dT) nucleic acid refers in particular to a nucleic acid molecule comprising a DNA having more than 90% T residues or comprising at least 10, at least 20, at least 25, at least 30 or at least 35 consecutive T residues. Respective capture oligonucleotides are well known and are commonly used to capture poly (A) nucleic acids such as poly (A) RNA. According to one embodiment, poly (dT) nucleic acids are used as capture oligonucleotides for separating poly (A) RNA, such as, in particular, poly (A) mRNA, which is a synthetic molecule with more than 90% nucleic acid bases T. As described above, analogs of nucleic acid bases T or U can also be used, as long as they can hybridize with A residues. Such capture oligonucleotides are also commonly referred to as oligo-dT and are well known in the art.

[0094] The capture probe can be present in the hybridization composition in a free form. The capture probe combined with the poly (A) nucleic acid can then be fixed to a solid phase during or after the hybridization reaction. The oligonucleotide can also be labeled by a compound that reacts with a second compound and fixed on a solid support in turn. Alternatively, the capture probe is provided in an immobilized form, wherein the capture probe is connected to a solid support. Preferably, a solid support functionalized with the capture probe is used, so it is included in the hybridization composition. The fixation of the solid to the solid support can be achieved using techniques well known in the art and standard. In some embodiments, the oligonucleotide is connected to a solid support using a linker structure.

[0095] The solid support may be provided by a variety of materials, including but not limited to: reaction vessels, microtiter plates, particles, magnetic particles, cellulose, columns, plates, membranes, filter papers and test strips, or any other solid support that can be used for separation techniques. Any support may be used as long as it is capable of liquid phase separation. Different solid supports are also used in known poly (A) nucleic acid separation methods. According to one embodiment, the solid support is provided by particles, also commonly referred to as beads. The particles used may be made of glass, silica, polymers, polystyrene-latex polymers, cellulose and / or plastic. According to a preferred embodiment, the solid support is provided by a suspension of particles functionalized with a capture probe. Magnetic particles are preferably used. When magnetic particles are used as solid supports, they may have superparamagnetic, paramagnetic, ferrimagnetic or ferromagnetic properties. Various magnetic particles can be easily separated by means of a magnetic field, for example by a permanent magnet, and therefore have advantages relative to processing. They are compatible with mature robotic systems capable of handling magnetic particles. Here, there are different robotic systems that can be used to handle magnetic particles combined with capture probes and poly (A) nucleic acid hybrids. According to one embodiment, magnetic particles are collected at the bottom or side of the reaction vessel, and the remaining liquid sample is removed from the reaction vessel, leaving the collected magnetic particles that are bound to the hybrid. The remaining sample can be removed by decantation or suction. In an alternative system, a magnet usually covered by a cover or envelope is immersed in the reaction vessel to collect the magnetic particles. In another selectable system, the sample containing the magnetic particles can be sucked into a pipette tip, and the magnetic particles can be collected in the pipette tip by applying to, for example, a magnet on the side of the pipette tip. The remaining sample can then be released from the pipette tip, and the collected magnetic particles that carry the hybrid are retained due to the magnet in the pipette tip. The collected magnetic particles can then be further processed. Such a system is also well known in the prior art, and is commercially available (e.g., BioRobot EZ1, Qiagen). Other processing systems are also known and can be used.

[0096] The particles may also be separated by filtration, centrifugation or by using a spin column which may be, for example, loaded with a particle suspension as is known to those skilled in the art. When the solid support is centrifuged it may settle or pass through a centrifugal filter device or column.

[0097] In some embodiments, the capture probe can be biotinylated or otherwise labeled to facilitate separation of hybrids. This embodiment is, for example, applicable to the use of capture oligonucleotides. Biotin can be derived from probe nucleotides using, for example, a linker, without weakening the ability of capture oligonucleotides to hybridize with poly (A) nucleic acids. Due to biotin and avidin / streptavidin reaction, avidin or streptavidin can be used in conjunction with biotinylated capture oligonucleotides. The avidin or streptavidin can be attached to a solid support, such as a particle or a container surface that can bind biotinylated capture oligonucleotides. The solid support can then be separated from the rest of the sample, such as by removing the solid support from the remaining sample, and vice versa, thereby separating the biotinylated capture oligonucleotides, which themselves are hybridized with poly (A) nucleic acids. The capture probe can also be labeled with a variety of different modifications known to those skilled in the art for separation. Non-limiting alternative methods include labeling the capture probe with an epitope tag and using an antibody or binding fragment thereof that recognizes the epitope for capture, for example, labeling an oligonucleotide with digoxigenin and capturing with an anti-digoxigenin antibody. In addition, haptens can be coupled to, for example, nucleotides or oligonucleotides. Common haptens for subsequent capture include biotin (biotin-11-dUTP), dinitrophenyl (dinitrophenyl-11-dUTP). These modifications include, for example, fluorescent modifications. Commercially available fluorescent nucleotide analogs that may be included include, but are not limited to: Cy3 TM -dCTP, Cy3 TM -dUTP, Cy TM 5-dCTP, fluorescein-12-dUTP, AlexaFluor®594-5-dUTP, AlexaFluor®-546-14-dUTP, etc. Fluorescent labeling can also be used as a separated part using commercially available anti-fluorescein antibodies. Also suitable are labels using radioisotopes, enzyme labels, and chemiluminescent labels.

[0098] In addition, in the case where the capture probe itself is not attached to the solid support, a hybridization binder immobilized on the solid support can be used to facilitate the separation of the formed hybrid, such as an anti-hybridization binder, such as an anti-DNA / RNA antibody or its binding fragment. Such an embodiment is suitable, for example, in the case where an RNA / DNA hybrid is formed when the capture probe hybridizes with a poly (A) nucleic acid. According to the above principles, various hybrid binders can be similarly immobilized on a solid support.

[0099] Therefore, many established systems are available, which achieve the hybrid formed between the capture probe and the poly (A) nucleic acid to be ultimately immobilized on a solid support, thereby facilitating the separation of the hybrid. As mentioned above, for ease of handling, it is preferred to use a solid support, such as particles functionalized with capture probes.

[0100] Incubation conditions

[0101] The hybridization composition is incubated under conditions for a sufficient time to allow the poly(A) nucleic acid to hybridize with the capture probe. As described above, a nucleic acid hybrid is formed between the poly(A) nucleic acid and the capture probe. During the incubation, the hybridization composition may be gently shaken or stirred.

[0102] In an embodiment, the hybridization composition is heated at a temperature of about 60°C to about 90°C, preferably about 65°C to about 75°C, before incubation under hybridization conditions. Each heating step helps to destroy the secondary structure of, for example, poly (A) nucleic acid, thereby making the poly (A) extension, such as the poly (A) tail, easy to hybridize with the capture probe. Each heating step can be performed for less than 10 minutes, less than 7 minutes, preferably less than 5 minutes. In an embodiment, for hybridization, the hybridization composition is incubated at a temperature of 50°C or less, 45°C or less, preferably 40°C or less, and more preferably at room temperature, so that the poly (A) RNA is hybridized with the capture probe. The corresponding incubation step can be performed for at least 4 minutes, preferably for a period of time in the range of 5 minutes to 30 minutes. The advantage of this method is that no long incubation is required, and the hybridization step, including the denaturation step (if performed), can be completed in less than 30 minutes, even less than 20 minutes. However, if necessary, longer incubation times can also be used.

[0103] After step (a) is completed, a nucleic acid hybrid is formed between the poly(A) nucleic acid and the capture probe.

[0104] Step (b)

[0105] In step (b), the formed hybrid is separated from the remaining sample, thereby separating the poly(A) nucleic acid from the sample and removing the poly(A) nucleic acid bound to the capture probe from the remaining sample.

[0106] As mentioned above, the separation of hybrid is preferably assisted by using a solid support that is combined with capture probes and / or the hybrid formed. The solid support that is combined with hybrids can be easily separated from the remaining sample. As mentioned above, according to the solid support used, the solid support can be removed from the remaining sample, or the remaining sample can be recovered and the solid support that is combined with hybrids can be left. Suitable solid supports and suitable separation procedures that can separate solid supports from the remaining sample are well-known, and are also described above in conjunction with capture probes, and with reference to the corresponding disclosures that this article is also applicable to. Suitable separation methods are also known and available to those skilled in the art.

[0107] Optional washing step (c)

[0108] In optional step (c), the isolated hybrids are washed one or more times. Even though this washing step (c) is optional, it is preferably performed to support the removal of unbound components and impurities that may interfere with certain downstream applications of the isolated poly(A) nucleic acid.

[0109] Therefore, according to a preferred embodiment, one or more washing steps are performed in step (c), thereby further purifying the isolated poly (A) nucleic acid. This can be conveniently performed, for example, when the hybrid is fixed on a solid support preferably provided by particles. Common washing solutions can be used, and suitable embodiments are known to those skilled in the art. Suitable washing solutions remove impurities, but do not substantially release the poly (A) nucleic acid from the hybrid and the loss of the poly (A) nucleic acid occurs during washing.

[0110] A washing solution having the same or lower concentration of sodium salt and / or having the same or lower concentration of quaternary ammonium salt compared to the hybridization solution used can be used as one or more washing solutions. If more than one washing buffer is used, the salt concentration can be reduced between washing steps.

[0111] When particles are used as a solid support for immobilizing capture probes, the particles can be resuspended by agitation, for example, by vortexing during washing.

[0112] Optional release step (d)

[0113] In an optional release step (d), the poly (A) nucleic acid is released from the hybrid. This can be conveniently performed, for example, when the hybrid is fixed on a solid support preferably provided by particles. In order to achieve release, one or more elution steps can be performed to elute the captured poly (A) nucleic acid.

[0114] Here, basically any release solution that can release the poly (A) nucleic acid from the hybrid and thus elute the bound poly (A) nucleic acid from the fixed support can be used, and this solid support is preferably used to assist the separation of the poly (A) nucleic acid. Various elution solutions that can effectively elute the poly (A) nucleic acid from the complementary capture probe are known to those skilled in the art and therefore do not need to be described in detail. Non-limiting examples include water, elution buffers, such as TE-buffers and low salt solutions, and their salt content is 150mM or less, 100mM or less, 75mM or less, 50mM or less, 25mM or less, 20mM or less, 15mM or less, 10mM or less or no salt. The elution solution may include, for example, a buffer, in particular, may include a biological buffer, such as Tris, MOPS, HEPES, MES, BIS-TRIS, propane, etc.

[0115] Elution may be aided by heating, for example to 50°C or higher, preferably 60°C or higher, more preferably 65°C or higher.

[0116] Elution can also be performed by shaking, which is particularly feasible when using a particulate solid support.

[0117] Furthermore, it is within the scope of the present invention to repeat the elution step to ensure that the captured poly(A) nucleic acids are effectively released. DETAILED DESCRIPTION

[0118] The specific preferred implementation of the method according to the first aspect is again as follows:

[0119] According to one embodiment, the method comprises:

[0120] (a) providing a hybridization composition comprising:

[0121] i) samples containing nucleic acids;

[0122] ii) a hybridization solution comprising:

[0123] aa. Sodium salt at a concentration ≤ 500 mM;

[0124] bb. Tetraalkylammonium salts as quaternary ammonium salts;

[0125] wherein the components of the hybridization solution may be added to the sample as a single solution or may be added to the sample separately in any order;

[0126] iii) a capture probe capable of hybridizing to a poly(A) stretch of a poly(A) nucleic acid;

[0127] The hybridization composition contains a hybridization solution sodium salt concentration of ≤250 mM,

[0128] and incubating the hybridization composition under conditions such that a nucleic acid hybrid is formed between the poly(A) nucleic acid and the capture probe;

[0129] (b) separating the formed hybrid from the remaining sample;

[0130] And preferably, also includes:

[0131] (c) washing and

[0132] (d) releasing the poly(A) nucleic acid from the hybrid.

[0133] According to one embodiment, the method comprises:

[0134] (a) providing a hybridization composition comprising:

[0135] i) samples containing nucleic acids;

[0136] ii) a hybridization solution comprising:

[0137] aa. Sodium salt, at a concentration selected from 50mM-350mM, 75mM-300mM, 100mM-250mM and 125mM-200mM;

[0138] bb. A tetraalkylammonium salt as a quaternary ammonium salt, having a concentration selected from 0.2M-2.5M, 0.5M-2M and 0.75M-1.5M;

[0139] Wherein, the components of the hybridization solution may be added to the sample as a single solution or may be added to the sample separately in any order;

[0140] iii) a capture probe capable of hybridizing to a poly(A) stretch of a poly(A) nucleic acid;

[0141] The hybridization solution sodium salt contained in the hybridization composition has a concentration selected from 25mM-175mM, 37.5mM-150mM, 50mM-125mM and 62.5mM-100mM; and the tetraalkylammonium salt has a concentration selected from 0.1M-1.25M, 0.25M-1M and 0.375M-0.75M;

[0142] and incubating the hybridization composition under conditions such that a nucleic acid hybrid is formed between the poly(A) nucleic acid and the capture probe;

[0143] (b) separating the formed hybrid from the remaining sample;

[0144] And preferably, it also includes:

[0145] (c) washing and

[0146] (d) releasing the poly(A) nucleic acid from the hybrid.

[0147] According to one embodiment, the method is used to separate poly(A) RNA from a total RNA sample, comprising:

[0148] (a) providing a hybridization composition comprising:

[0149] i) samples containing nucleic acids;

[0150] ii) a hybridization solution comprising:

[0151] aa. Sodium chloride as a sodium salt, at a concentration selected from 75mM-250mM, 100mM-200mM, and 125mM-175mM;

[0152] bb. a tetraalkylammonium salt selected from the group consisting of tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB) as a quaternary ammonium salt, wherein the concentration is selected from 0.25M-2.5M, 0.5M-2M and 0.75M-1.5M;

[0153] Wherein, the components of the hybridization solution may be added to the sample as a single solution or may be added to the sample separately in any order;

[0154] iii) a capture probe capable of hybridizing to a poly(A) stretch of a poly(A) nucleic acid, wherein the capture probe is immobilized on a solid support, preferably the solid support is provided by particles;

[0155] The hybridization solution sodium salt contained in the hybridization composition has a concentration selected from 37.5mM-125mM, 50mM-100mM, 62.5mM-87.5mM and 62.5mM-100mM; and the tetraalkylammonium salt has a concentration selected from 0.125M-1.25M, 0.5M-1M and 0.375M-0.75M,

[0156] and incubating the hybridization composition under conditions such that a nucleic acid hybrid is formed between the poly(A) nucleic acid and the capture probe;

[0157] (b) separating the formed hybrid bound to the solid support from the remaining sample;

[0158] And preferably, it also includes:

[0159] (c) washing and

[0160] (d) releasing the poly(A) nucleic acid from the hybrid.

[0161] Application and use of the method according to the first aspect

[0162] The method according to the first aspect can effectively separate poly(A) nucleic acids from various samples while minimizing the carryover of non-poly(A) nucleic acids in the separated poly(A) nucleic acids.

[0163] As used herein, the term "nucleic acid" or "nucleic acids" refers specifically to a polymer including ribonucleic acids and / or deoxyribonucleic acids covalently bonded, in particular, by phosphodiester bonds (in some cases phosphorothioate, methylphosphonate, etc.) between subunits. The term encompasses naturally occurring nucleic acids as well as synthetic nucleic acids.

[0164] As mentioned above, the term "poly (A) nucleic acid" and its corresponding terms specifically refer to a single-stranded poly (A) stretch containing continuous adenine base ("A") residues. Such a poly (A) stretch is usually provided at the 3' end of the nucleic acid, and is then also referred to as a poly (A) tail. The term "poly (A) nucleic acid" particularly includes any nucleic acid species containing a poly (A) tail at its 3' end, in particular a poly (A) tail-containing sequence having at least 10, at least 15 or at least 20 A residues. The term poly (A) nucleic acid refers to poly (A) RNA as well as poly (A) DNA. As described herein, the poly (A) nucleic acid is preferably a poly (A) RNA, in particular a poly (A) mRNA. Poly (A) RNA is the main polyadenylated nucleic acid of interest in research and diagnostic applications.

[0165] Due to the favorable hybridization conditions, non-poly (A) nucleic acids are not substantially captured by the method of the present invention. Non-poly (A) nucleic acids, i.e., nucleic acids without single-stranded poly (A) extensions (e.g., poly (A) tails), are not captured and thereby effectively removed during the separation process of the present invention, including, for example, non-polyadenylated RNAs, such as rRNA, tRNA, snRNA, snoRNA, and immature portions of mRNA lacking poly (A). The term non-poly (A) nucleic acids may also refer to other non-polyadenylated nucleic acids, such as non-polyadenylated DNA (if present in the sample).

[0166] The term "sample containing nucleic acid" is used in a broad sense in this article, and is intended to include multiple sources and compositions containing nucleic acid. The sample containing nucleic acid can be derived from a biological sample, but the term also includes other, for example, an artificial sample comprising poly (A) nucleic acid (such as provided in vitro from the nucleic acid of the poly (A) tail). Particularly refers to a composition containing the purified nucleic acid separated from a biological sample (for example, using the inventive method to separate the total RNA of poly (A) nucleic acid). However, the sample containing nucleic acid can also be provided by a biological sample, particularly a lysate of a biological sample. As known to those skilled in the art, poly (A) nucleic acid, for example poly (A) RNA, can be separated from the sample lysate.

[0167] Exemplary biological samples include, but are not limited to, cell samples, environmental samples, samples obtained from the body, in particular body fluid samples, and human, animal or plant tissue samples. Non-limiting examples include, but are not limited to, cells, whole blood, blood products, red blood cells, white blood cells, buffy coat, plasma, serum, swabs, urine, sputum, saliva, semen, lymph, amniotic fluid, cerebrospinal fluid, peritoneal effusion, pleural effusion, biopsy samples, cyst fluid, synovial fluid, vitreous fluid, aqueous humor, bursa, eye wash, eye aspirate, plasma, serum, lung lavage fluid, lung aspirate, animal (especially human) or plant tissue, including but not limited to liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, cell culture, and lysate, extract or material and part obtained from the above samples. Preferably, the sample is derived from a biological sample of a human, animal or plant. The sample can be selected from the group consisting of cells, tissues, tumor cells and body fluids, such as blood, blood products such as buffy coat, plasma and serum, urine, body fluids, sputum, feces, CSF and sperm, epithelial swabs, biopsies, bone marrow samples and tissue samples, preferably organ tissue samples. The term "sample" also includes processed samples, such as preserved, fixed and / or stable samples. As mentioned above, the nucleic acid-containing sample included in the hybridization composition is preferably provided by nucleic acid purified from the corresponding sample, such as total RNA. However, the nucleic acid-containing sample can also be a crude sample comprising a poly (A) nucleic acid in a released form, and can be provided by a lysate obtained by a corresponding biological sample.

[0168] According to a preferred embodiment, the sample containing nucleic acid is a purified nucleic acid sample. Preferably, the sample containing nucleic acid is total RNA, and poly (A) RNA is isolated from the total RNA using the method of the present invention. Total RNA can be isolated from various samples, for example using any common RNA purification method. Suitable methods are well known in the prior art and therefore do not need to be described in detail here. Suitable methods include, but are not limited to: using a phenol / chloroform-based method to separate RNA, using a chaotropic agent, alcohol and a solid phase (for example, particularly a silicon-containing solid phase (for example, silicon dioxide, glass fiber, silicon carbide)) to separate RNA, alcohol precipitation, precipitation by other organic solvents, polymers or cationic detergents, etc.

[0169] According to one embodiment, the nucleic acid sample containing poly (A) nucleic acid, preferably poly (A) RNA, is a biological sample processed, such as a cracked sample. Any suitable cracking method compatible with the separation of poly (A) nucleic acid can be used to crack the sample. Various methods for cracking biological samples are known to those skilled in the art, and also depend on the specific sample type to be processed. Cracking can be, for example, based on or including chemical or mechanical cracking procedures, and non-limiting examples include cracking methods involving lysing agents, such as detergents, proteolytic enzymes or chaotropic salts, heating, ultrasound, mechanical crushing, etc. According to one embodiment, a lysate having removed DNA is used as a sample containing nucleic acid. DNA can be removed from the lysate, for example, by performing DNase digestion or by selective separation and removing DNA from the lysate therefrom. Suitable methods for selectively combining and removing DNA therefrom are as described in EP 0880537 and WO 95 / 21849, which are incorporated herein by reference. For example, if in the absence of short-chain alcohols (such as ethanol or isopropanol), then use a chaotropic agent such as a chaotropic salt to crack the sample, it is possible to establish selective binding conditions for DNA, especially if a silicon-containing solid phase is used. If desired, the combined DNA (such as further processed, such as sequenced) can be further used, so that for example, optionally washing and eluting from the nucleic acid-bound solid phase can be provided, thereby providing a DNA portion that is substantially free of RNA. However, if it is not DNA that is of interest, i.e., if only RNA is of interest, it is also possible to simply discard the combined DNA. In addition, the lysate containing RNA can be removed before the poly (A) RNA is separated therefrom, to remove such as cell debris and other contaminants.

[0170] The method of the present invention is particularly suitable for isolating poly(A) RNA from eukaryotic samples. The method effectively removes a large number of rRNAs, such as 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA, mitochondrial 12S rRNA and mitochondrial 16S rRNA. As described, effective removal of such unwanted RNA is essential, particularly for next generation sequencing applications, otherwise valuable sequencing capacity will be wasted.

[0171] In experiments using the method according to the present invention, more than 99% of non-poly (A) RNA such as 5S rRNA, 5.8S rRNA and 28S rRNA were removed. Therefore, according to one embodiment, the amount of non-poly (A) RNA in the isolated poly (A) RNA is ≤3%, ≤2%, ≤1.5%, preferably ≤1%, ≤0.75%, ≤0.5%, ≤0.25%, ≤0.15%, ≤0.1%, ≤0.05%. As mentioned above, the non-poly (A) RNA to be removed is rRNA. The typical rRNA that needs to be effectively removed is 5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA. In addition, 12mt and 16mt rRNA can also be effectively removed using the method of the present invention.

[0172] The method can be used for the preparation of poly(A) nucleic acids for any purpose, which conventionally requires the isolation of poly(A) nucleic acids, particularly poly(A) RNA. Non-limiting examples include, but are not limited to, the isolation of poly(A) RNA from total RNA or directly from biological samples such as lysates of cells and tissues, for cDNA synthesis, cDNA library construction, amplification based on methods such as reverse transcription PCR, subtractive hybridization, polyadenylated in vitro transcripts, direct isolation of oligonucleotides or other nucleic acids, in vitro translation, SAGE technology, expression analysis, expression array and expression chip analysis, microarray analysis, ribonuclease and S1 nuclease protection, primer extension, RNA northern, dot and slot blotting, microinjection, and in addition, for sequencing applications, such as, in particular NGS applications. It can also be used to selectively remove poly(A) nucleic acids from samples in the case where poly(A) nucleic acids are not needed and should be removed from the sample. In this case, the washing and elution steps for further purification and isolation of poly(A) nucleic acids can be eliminated. However, it may also be of interest to analyse the isolated and removed poly(A) nucleic acids separated from the non-poly(A) nucleic acids, so in this case they may be washed and eluted.

[0173] The method of the present invention is particularly advantageous because it is highly effective, thereby minimizing the loss of poly(A) nucleic acids by means of the separation process, is highly selective for poly(A) nucleic acids, thereby reducing the residue of unwanted non-poly(A) nucleic acids in the separated poly(A) nucleic acid fraction, and furthermore, requires minimal processing time. Favorable results can be achieved after one separation cycle, which is a considerable advantage over prior art methods, which often require at least two enrichment cycles to provide sufficiently pure poly(A) nucleic acids. Therefore, the method is very suitable for processing multiple samples simultaneously in high-throughput applications.

[0174] As mentioned above, the method according to the present invention is particularly suitable for preparing poly (A) RNA for next generation sequencing (NGS) applications (such as transcriptome sequencing). For such applications, it is very important to effectively capture poly (A) RNA and remove non-poly (A) RNA (such as a large amount of rRNA) from the poly (A) RNA of interest during the separation process, because the more non-interested RNA (such as rRNA) is reduced, the more information can be obtained from a sequencing run. The present invention provides such a method, and thus has made an important contribution to the art. For this purpose, preferably purified total RNA is used as a sample material containing nucleic acids. The eluent rich in poly (A) RNA obtained after the separation process can be used to construct a sequencing library. This embodiment will also be further explained in detail in conjunction with the method of the second aspect, and reference is made to the corresponding disclosure.

[0175] Method for sequencing poly(A) nucleic acid

[0176] According to a second aspect, there is provided a method for sequencing a poly(A) nucleic acid, preferably a poly(A) RNA, comprising:

[0177] (a) isolating poly(A) nucleic acid from a sample containing nucleic acid using the method according to the first aspect;

[0178] (b) Sequencing the isolated poly(A) nucleic acid molecules.

[0179] In step (a), the method according to the first aspect is performed to isolate poly(A) nucleic acids from the sample. Details of the method and the associated advantages are as described above, and we also refer to the corresponding disclosures applicable here. Preferably, poly(A) RNA is isolated as poly(A) nucleic acid. Preferably, poly(A) RNA is isolated from a total RNA sample according to steps (a) and (b) of the method of the first aspect, and washing and elution are performed according to steps (c) and (d) of the above method of the first aspect.

[0180] The purified poly (A) nucleic acid is then sequenced in step (b). According to one embodiment, the sequencing comprises preparing a sequencing library from the isolated poly (A) nucleic acid. Preferably, the sequencing library is suitable for large-scale parallel sequencing, and the sequencing comprises sequencing the molecules contained in the library in parallel. Individual sequencing libraries are known in the art. The sequencing library may comprise a plurality of double-stranded molecules, and is preferably suitable for large-scale parallel sequencing, and is therefore suitable for next generation sequencing. The preparation of the corresponding sequencing library is also the current standard in transcriptome sequencing. The plurality of double-stranded nucleic acid molecules present in the sequencing library may be linear or circular, and preferably, the nucleic acid molecules contained in the sequencing library are linear. Sequencing libraries suitable for next generation sequencing may be prepared using methods known in the prior art. Preferably, the double-stranded molecules in the sequencing library are DNA molecules. For this purpose, in the case where the poly (A) nucleic acid is poly (A) RNA, the poly (A) RNA may be reverse transcribed into cDNA. Typically, the method for preparing a sequencing library suitable for next-generation sequencing includes obtaining DNA fragments, optionally followed by DNA repair and end finishing, and finally, typically NGS platform-specific adapter ligation. According to one embodiment, the cDNA obtained can be fragmented, for example, by shearing (e.g., ultrasound, hydraulic shearing, ultrasound, atomization, or enzymatic cleavage), thereby providing DNA fragments suitable for subsequent sequencing. However, fragmentation to the desired length may occur at the RNA level, and therefore is before cDNA synthesis. For example, the isolated poly (A) RNA can be fragmented by magnesium-catalyzed hydrolysis of RNA. The length of the fragment can be selected based on the sequencing capability of the next-generation sequencing platform for subsequent sequencing. Typically, the fragment obtained has a length of 1500bp or less, 1000bp or less, 750bp or less, 600bp or less, preferably 500bp or less, because this corresponds to the sequencing capability of most current next-generation sequencing platforms. The fragmented DNA can be repaired and end-finished again using methods known in the prior art, thereby providing such as blunt ends or nucleotide overhangs, such as A overhangs.

[0181] In addition, the adapter can be connected to the 5' and / or 3' ends of the DNA fragment, preferably at both ends of the obtained fragment. The specific design of the adapter depends on the next generation sequencing platform to be used and the purpose of the present invention, and basically any adapter for preparing the next generation sequencing library can be used. Therefore, the sequencing library can contain or consist of randomly fragmented double-stranded DNA molecules, which are connected to the adapter sequence at their 3' and 5' ends. The adapter provides a known sequence, thereby providing a known template for amplification and / or sequencing primers. As an adapter, a double-stranded or partially double-stranded nucleic acid of a known sequence can be used. The adapter can have a sticky end with a blunt end, a 3' or 5' overhang, and can be provided by a Y-shaped adapter or a stem ring adapter (see US 2009 / 0298075). The Y-shaped adapter is as described in (see US 7,741,463). Optionally, the adapter can also provide a separate index, so that two or more sequencing libraries can be subsequently pooled before sequencing. As discussed, sequencing is preferably performed on a next generation sequencing platform. In NGS, sequencing is usually performed by repeated cycles of polymerase-mediated nucleotide extension, or in a common form, by iterative cycles of oligonucleotide ligation. After obtaining a sequencing library using the method of the present invention, clonal isolation and subsequent amplification of single molecules are performed by in vitro template preparation reactions such as emulsion PCR (pyrophosphate sequencing from Roche454, semiconductor sequencing from Ion Torrent, SOLiD sequencing by ligation from Life Technologies, sequencing by synthesis from Intelligent Biosystem), bridge amplification of flow cells (e.g. Solexa / Illumina), isothermal amplification by Wildfile technology (Life Technologies), or circular clones (rolonies) / nanospheres (Complete Genomics, Intelligent Biosystems, Polonator) produced by rolling circle amplification. Sequencing technologies such as Heliscope (Helicos), SMRT technology (Pacific Biosciences) or nanopore sequencing (Oxford Nanopore) can directly sequence single molecules without prior clonal amplification. Sequencing can be performed on any corresponding platform using sequencing libraries prepared from isolated poly(A) RNA. Suitable methods for preparing sequencing libraries and next generation sequencing methods are described in Metzker, 2011, Voelkerding, 2009 and WO12 / 003374.

[0182] The advantages of using the method of the first aspect to prepare poly(A) nucleic acid (such as poly(A) RNA in step (a)) and the corresponding sequencing results are as described above, and reference is made to the corresponding disclosure.

[0183] Hybridization Solutions and Kits

[0184] According to a third aspect, there is provided an aqueous hybridization solution comprising:

[0185] aa. Sodium salt at a concentration ≤ 500 mM;

[0186] bb. Quaternary ammonium salt.

[0187] The hybridization solution according to the third aspect can be combined with and perform the methods of the first, second and fourth aspects of the invention, and it can be used in particular to establish favorable binding conditions for capturing poly(A) nucleic acids, or it can be used to provide stringent washing conditions that help remove non-poly(A) nucleic acids that may have been bound during the capture step, for example without using favorable hybridization conditions according to the invention (i.e., wherein a low concentration of sodium salt and a quaternary ammonium salt (such as preferably a tetraalkylammonium salt)) are used in combination.

[0188] The hybridization solution, in particular suitable and preferred hybridization solution components and hybridization solution component concentrations and suitable and preferred mixing ratios with the sample (or diluted sample) are described in detail above in conjunction with the method of the first aspect of the present invention. Reference is made to the above disclosure, which also applies here. Subsequently, a brief description of non-limiting selected embodiments will be given again.

[0189] The hybridization solution may contain a sodium salt concentration of ≤500mM. The sodium salt is preferably a sodium halide, more preferably sodium chloride. The hybridization solution may contain a sodium salt concentration selected from the following ranges: 50mM-500mM, 75mM-400mM, 85mM-350mM, 100mM-300mM, 115mM-250mM, 120mM -225mM and 125mM-200mM. According to one embodiment, the hybridization solution contains a sodium salt concentration selected from the range of 125mM-175mM. As demonstrated in the examples, this hybridization solution provides particularly good results when contacted with an equal volume of sample (or diluted sample).

[0190] The hybridization solution may contain a quaternary ammonium salt concentration of ≤6M, ≤5M, ≤4M or ≤3M. The hybridization solution may contain a quaternary ammonium salt concentration of ≥200mM, ≥250mM, ≥500mM or ≥750mM. Preferably, the hybridization solution contains a quaternary ammonium salt concentration selected from 0.2M-3.5M, 0.25M-3M, 0.5M-2.5M, 0.75M-2M and 0.75M-1.5M. The quaternary ammonium salt is preferably a tetraalkylammonium salt, such as tetramethylammonium salt (TMA) or tetraethylammonium salt (TEA). Suitable tetraalkylammonium salts include, but are not limited to, tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetraethylammonium nitrate (TEAN), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB). According to one embodiment, the quaternary ammonium salt is not tetramethylammonium sulfate. Preferably, the hybridization solution contains tetramethylammonium bromide as the quaternary ammonium salt.

[0191] According to one embodiment, the hybridization solution comprises one or more compounds selected from the group consisting of detergents, chelating agents and buffers. The details are described above in conjunction with the first aspect and reference is made to the above disclosure. According to one embodiment, the hybridization solution does not comprise chaotropic ions.

[0192] As mentioned above, the hybridization conditions used in the inventive method are based on the balanced combination of sodium salt and quaternary ammonium salt, which has caused the effective separation of poly (A) nucleic acid, and has prevented the residual of non-poly (A) nucleic acid simultaneously.Therefore, except sodium salt and quaternary ammonium salt, hybridization solution does not comprise other hybridization promoting salts of the concentration that will offset these beneficial effects.Therefore, according to embodiments, hybridization solution does not comprise hybridization promoting salts, for example lithium chloride or potassium chloride or other non-sodium halides, MgCl2 and / or chaotropic salts of the concentration that will offset the beneficial effects obtained by sodium salt and quaternary ammonium salt combination.In embodiments, if present in hybridization solution, the concentration of these salts is 100mM or lower, 75mM or lower, 50mM or lower or 25mM or lower.Preferably, described hybridization solution does not contain any hybridization promoting salt except sodium salt and quaternary ammonium salt.

[0193] According to one embodiment, the hybridization solution comprises a sodium salt at a concentration of ≤500 mM and a tetraalkylammonium salt as a quaternary ammonium salt. The hybridization solution may comprise a sodium salt at a concentration of ≥50 mM, ≥75 mM or ≥100 mM. The hybridization solution may comprise, for example, a sodium salt at a concentration selected from 50 mM-350 mM, 100 mM-300 mM, 115 mM-250 mM, 120 mM-225 mM and 125 mM-200 mM, and a tetraalkylammonium salt as a quaternary ammonium salt at a concentration selected from 0.5 M-2.5 M, 0.75 M-2 M and 0.75 M-1.5 Mg. For example, the hybridization solution may contain sodium chloride at a concentration selected from 75 mM to 250 mM, 100 mM to 200 mM and 125 mM to 175 mM, and a tetraalkylammonium salt selected from tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetraethylammonium chloride, tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB) as a quaternary ammonium salt at a concentration selected from 0.5 M to 2.5 M, 0.75 M to 2 M and 0.75 M to 1.5 M. As mentioned above, the use of tetraethylammonium bromide (TEAB) is particularly preferred.

[0194] The hybridization solution according to the third aspect can be advantageously used in the preparation of the above-mentioned hybridization composition in combination with the method according to the first aspect. With reference to the disclosure above, it is also applicable here. In addition, it can be used to establish stringent washing conditions, which are described in detail below.

[0195] According to a fourth aspect, there is provided a kit for isolating poly(A) nucleic acid from a sample, comprising:

[0196] (a) the hybridization solution according to the third aspect;

[0197] (b) A capture probe capable of hybridizing to the poly(A) stretch of the poly(A) nucleic acid.

[0198] The hybridization solution according to the third aspect is as described above, and reference is made to the above disclosure. Details and preferred embodiments of the capture probe are also described in conjunction with the method of the first aspect, and reference to the disclosure applies equally hereto. As described above, the capture probe can be a capture oligonucleotide, such as a preferably synthetic nucleic acid molecule containing oligo(T) or oligo(U) or a mixture thereof, or any other suitable or fixable capture probe to a solid support. Suitable and preferred embodiments of the solid support are also described in conjunction with the method of the first aspect, and reference is made to the above disclosure and applies equally hereto. As described above, according to one embodiment, the capture probe is bound to a non-magnetic or magnetic particle.

[0199] The kit is particularly suitable for the method described in the first aspect. In addition, the kit may contain instructions for use and / or information. For example, the kit may contain instructions and / or information about using a certain volume of hybridization solution and a certain volume of nucleic acid-containing sample and / or diluent solution (such as water) to achieve an effective concentration of sodium salt and quaternary ammonium salt in the hybridization solution. Depending on the volume / ratio used, different salt concentrations can be used in the hybridization solution.

[0200] Method for washing poly(A) nucleic acid hybrids

[0201] The inventors have also found that the favorable hybridization conditions using a low concentration of sodium salt in combination with a quaternary ammonium salt not only provide highly stringent and selective capture conditions for poly (A) nucleic acids, but also provide highly stringent washing conditions. Therefore, these hybridization conditions can be advantageously used to remove non-poly (A) nucleic acids during the washing step.

[0202] Therefore, according to a fifth aspect, there is provided a method for isolating a poly(A) nucleic acid having a single-stranded poly(A) stretch from a sample containing nucleic acid, comprising:

[0203] (a) hybridizing a poly(A) nucleic acid with a capture probe capable of hybridizing to a poly(A) stretch of the poly(A) nucleic acid, thereby forming a nucleic acid hybrid between the poly(A) nucleic acid and the capture probe;

[0204] (b) separating the formed hybrids from the remaining sample;

[0205] (c) washing the separated hybrids using the hybridization solution of the third aspect, wherein the components of the hybridization solution may be added to the hybrids as a single solution, or may be added to the hybrids separately in any order to produce the hybridization solution for washing;

[0206] (d) Releasing poly(A) nucleic acid from the washed hybrids.

[0207] In step (a), the poly (A) nucleic acid is hybridized with a capture probe capable of hybridizing with a poly (A) extension of the poly (A) nucleic acid to form a nucleic acid hybrid. As described above, a capture oligonucleotide comprising a complement to the poly (A) tail can be used. In the method of the fifth aspect, any method and its hybridization conditions can be used to provide a corresponding hybrid. Therefore, methods of the prior art can also be used. However, it is preferred that the above-mentioned hybridization conditions are combined with the method of the first aspect and are also used in step (a) of the fifth aspect method. Details about the poly (A) nucleic acid and the capture probe are described in conjunction with the first aspect of the present invention. The corresponding disclosure also applies here.

[0208] In step (b), the formed hybrid is separated from the remaining sample. Here, any common separation technique can be used. The exemplary embodiment is described above in conjunction with step (b) of the first aspect method. The same disclosure is cited here.

[0209] In step (c), the hybridization solution of the third aspect is used to wash the separated hybrid. The details and preferred embodiments of the corresponding hybridization solution are as described above, and reference is made to the corresponding disclosure and is applicable hereto. The components of the hybridization solution can be a single solution added for washing (preferably), or can be added separately to the hybrid in any order to produce a hybridization solution for washing. The hybridization solution can also be diluted with a suitable diluent solution, such as water or other preferred solvents. For the ratio of hybridization solution: diluent solution, the same ratio of hybridization solution: sample (or diluted sample) as in the above-mentioned first aspect method can be used. According to one embodiment, hybridization conditions are established during washing and are therefore used in a washing composition comprising a hybridization solution and a hybrid, which corresponds to the above-mentioned hybridization conditions of the hybridization composition of the first aspect method. Reference is made to the corresponding disclosure, which is also applicable hereto. Non-limiting embodiments are as required below:

[0210] Subsequently, suitable and preferred embodiments of washing compositions and hybridization solutions suitable for forming these washing conditions are disclosed. Due to the addition of a hybridization solution and an optional diluent solution, a washing composition is formed.

[0211] When describing and defining the concentrations of components of a "hybridization solution" in a wash composition, the volumes contributed by the capture probes and solid supports are not taken into account when determining the concentrations of the components described subsequently in the wash composition. According to one embodiment, the hybridization solution comprises the components in concentrated form, which can be diluted with a diluent solution to achieve an appropriate final concentration in the wash composition for washing hybrids.

[0212] The hybridization solution for washing and the washing composition comprises a sodium salt. This also includes using a mixture of different sodium salts as the "one" sodium salt. The sodium salt promotes the binding of poly (A) nucleic acid to the capture probe, thereby reducing the risk of loss of poly (A) nucleic acid during washing. It can be an inorganic or organic sodium salt. According to one embodiment, the sodium salt is a sodium halide. According to one embodiment, the sodium salt is not a chaotropic salt. Preferably, the sodium halide is sodium chloride.

[0213] With regard to non-poly (A) nucleic acid contaminants that need to be reduced, it has been found that reducing the concentration of sodium salt in the washing composition is beneficial. Therefore, according to one embodiment, the washing composition comprises a hybridization solution having a sodium salt concentration of ≤250 mM. The washing composition comprises a hybridization solution having a sodium salt concentration selected from 25 mM-250 mM, 35 mM-200 mM, 40 mM-175 mM, 50 mM-150 mM, 55 mM-125 mM, 60 mM-115 mM and 60 mM to 100 mM.

[0214] According to a preferred embodiment, the washing composition comprises a sodium salt concentration of ≤200 mM, ≤175 mM, ≤150 mM, ≤125 mM or ≤100 mM of the hybridization solution. Such lower concentrations of sodium salt in the washing composition are preferred because they provide stringent conditions for specific hybridization of poly(A) nucleic acids to capture probes, while non-specifically hybridized non-poly(A) nucleic acids are washed away.

[0215] The hybridization solution and its washing composition contain a quaternary ammonium salt. This also includes using a mixture of different quaternary ammonium salts as a "mono" quaternary ammonium salt. According to one embodiment, the quaternary ammonium salt is a tetraalkylammonium salt. The tetraalkylammonium salt can be a tetramethylammonium salt (TMA) or a tetraethylammonium salt (TEA). Suitable tetraalkylammonium salts include, but are not limited to, tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetraethylammonium nitrate (TEAN), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB). According to one embodiment, the quaternary ammonium salt is not tetramethylammonium sulfate. Preferably, tetramethylammonium bromide is used as the quaternary ammonium salt.

[0216] According to one embodiment, the washing composition comprises a quaternary ammonium salt concentration of ≤3M, ≤2.5M, ≤2M or ≤1.5M in the hybridization solution. The washing composition may comprise a quaternary ammonium salt concentration of ≥100mM, ≥125mM, ≥250mM or ≥375mM in the hybridization solution. Preferably, the washing composition comprises a quaternary ammonium salt concentration selected from 0.1M-1.75M, 0.125M-1.5M, 0.25M-1.25M, 0.375M-1M and 0.375M-0.75M. As described above, the quaternary ammonium salt is preferably a tetraalkylammonium salt, and suitable examples are as described above.

[0217] According to one embodiment, the hybridization solution used to establish conditions in the detergent composition comprises a quaternary ammonium salt concentration of ≤6M, ≤5M, ≤4M or ≤3M. The hybridization solution may comprise a quaternary ammonium salt concentration of ≥200mM, ≥250mM, ≥500mM or ≥750mM. It may comprise a quaternary ammonium salt concentration selected from 0.2M-3.5M, 0.25M-3M, 0.5M-2.5M, 0.75M-2M and 0.75M-1.5M.

[0218] One skilled in the art can determine appropriate concentrations of sodium salts and quaternary ammonium salts based on the teachings provided herein.

[0219] Non-limiting preferred embodiments of the wash composition, particularly with respect to the sodium salt (preferably sodium chloride) and quaternary ammonium salt (preferably tetraalkylammonium salt) contained are described below. As described above, when describing the concentration of the components of the hybridization solution in the wash composition, the volume contributed by the capture probes and the device used to assist in separation (such as a solid support (if used to assist in capture and separation)) is not taken into account for the purpose of determining the concentration of the components of the wash composition. In addition, as described above, the components of the hybridization solution can be added to the sample as a single solution, or can be added separately to the separated hybrids in any order, for example, using two or more solutions containing at least one hybridization solution chemical to produce a hybridization solution for washing.

[0220] According to one embodiment, the washing composition comprises a sodium salt of the hybridization solution at a concentration of ≤250 mM, and a tetraalkylammonium salt as a quaternary ammonium salt. According to one embodiment, the washing composition comprises a sodium salt of the hybridization solution at a concentration selected from 25 mM-175 mM, 50 mM-150 mM, 55 mM-125 mM, 60 mM-115 mM and 60 mM-100 mM, and a tetraalkylammonium salt as a quaternary ammonium salt at a concentration selected from 0.25 M-1.25 M, 0.375 M-1 M and 0.375 M-0.75 M.

[0221] According to one embodiment, the washing composition comprises a sodium salt in the hybridization solution at a concentration selected from 37.5mM-125mM, 50mM-100mM and 55mM-87.5mM and 60mM-100mM, and a tetraalkylammonium salt as a quaternary ammonium salt at a concentration selected from 0.25M-1.25M, 0.375M-1M and 0.375M-0.75M, wherein the sodium salt is sodium chloride.

[0222] As mentioned above, hybridization conditions of the present invention that can also be used for stringent washing conditions are based on the balanced combination of sodium salt and quaternary ammonium salt, which has caused the effective separation of poly (A) nucleic acid, while preventing the residual of non-poly (A) nucleic acid. Therefore, except sodium salt and quaternary ammonium salt, described hybridization solution and cleaning composition thereof do not comprise other hybridization promoting salts of the concentration that will offset these beneficial effects. Therefore, according to embodiments, described hybridization solution and / or cleaning composition do not contain hybridization promoting salts, such as lithium chloride or potassium chloride or other non-sodium halides, MgCl2 and / or chaotropic salts of the concentration that will offset the beneficial effects obtained by the combination of sodium salt and quaternary ammonium salt. In embodiments, the concentration of these salts (if present) is 100mM or lower, 75mM or lower, 50mM or lower or 25mM or lower. Preferably, described hybridization solution does not contain any hybridization promoting salt except sodium salt and quaternary ammonium salt.

[0223] In step (d), the poly(A) nucleic acid is released from the washed hybrid. The details of the release step (d) have been described above in conjunction with the method of the first aspect. Reference is made to the above disclosure, which also applies here.

[0224] The use of the washing conditions disclosed herein in at least one washing step in the poly(A) nucleic acid isolation procedure has the following advantages: non-poly(A) nucleic acids bound in the hybridization step (a) (e.g., without the hybridization conditions of the present invention) can be subsequently washed away. In addition, one or more additional washing steps can be performed. This is also preferred in order to improve the purity of the poly(A) nucleic acids and remove hybridization solution components that may potentially interfere with downstream applications.

[0225] As mentioned above, the poly(A) nucleic acid is preferably poly(A) RNA. Reference is made to the above disclosure in conjunction with the method of the first aspect, which also applies here.

[0226] The present invention is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used for the practice or testing of embodiments of the present invention. Numerical ranges include numbers within the defined ranges. The titles provided herein are not limitations on various aspects or embodiments of the present invention, and they can be read by reference to the entire specification.

[0227] As used in this specification and claims, the singular forms "a", "an", and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a sodium salt" includes a single type of sodium salt, as well as two or more sodium salts. Similarly, reference to a "quaternary ammonium salt", a "capture probe", "detergent", a "buffer", etc., includes single entities and combinations of two or more such entities. References to "the present disclosure" and "the present invention", etc., include single or multiple aspects taught herein; and so on. Aspects taught herein are encompassed by the term "invention".

[0228] The term "solution" as used herein refers in particular to a liquid composition, preferably an aqueous composition. It may be a homogeneous mixture with only a single phase, but it is also within the scope of the present invention that the solution contains solid components, such as a precipitate.

[0229] According to one embodiment, the subject matter described herein, including certain steps in the case of a method, or containing certain ingredients in the case of a composition, solution and / or buffer, refers to the subject matter consisting of the corresponding steps or ingredients. Preferably, the preferred embodiments described herein are selected and combined, and the specific subject matter generated from the corresponding combination of the preferred embodiments also belongs to the present disclosure.

[0230] Example

[0231] The examples are for illustrative purposes only and should not be construed as limiting the present invention in any manner.

[0232] Materials and methods

[0233] Isolation of poly(A) nucleic acids

[0234] In this experiment, total RNA isolated from Jurkat cells was used as the nucleic acid-containing sample for isolating poly(A) RNA. As the capture probe, dC covalently coupled to the surface (non-magnetic) of polystyrene latex particles (Oligotex suspension; Qiagen) was used. 10 T 30 Capture oligonucleotides or dT covalently coupled to the surface of magnetic particles (Seradyn magnetic beads) 14 Capture oligonucleotides.

[0235] Basic Plan

[0236] The poly(A) nucleic acid isolation protocol performed was based on the Oligotex® manual protocol (Qiagen). The main steps of the “basic protocol” are as follows:

[0237] - Add nucleic acid-containing samples (total RNA) to an RNase-free reaction tube and adjust the volume to 250 μl with RNase-free water.

[0238] - Add 250 μl of hybridization solution.

[0239] - Add capture oligonucleotides immobilized on a solid support (15 μl of Oligotex suspension (Qiagen) or 25 μl of functionalized Seradyn magnetic beads if not otherwise stated).

[0240] - Mix the contents and incubate at 70 °C for 3 minutes to denature the RNA.

[0241] - Incubate at 20°C-30°C for 10 minutes to allow hybridization of poly(A) RNA to the capture oligonucleotide.

[0242] - Separation of the formed hybrids from the remaining sample. The separation process depends on the type of solid support used. In the case of Oligotex particles, the sample is centrifuged to precipitate the Oligotex / poly(A)RNA complexes and the supernatant (remaining sample) is discarded. In the case of magnetic particles, the magnetic particles / poly(A)RNA complexes are precipitated with the aid of a magnetic field and the supernatant (remaining sample) is discarded. Alternatively, separation can also be performed using spin columns.

[0243] - Resuspend the precipitated complexes in 400 μl of washing buffer (10 mM Tris-Cl, pH 7.5; 150 mM NaCl; 1 mM EDTA) by vortexing. In the case of Oligotex particles, add the resuspended sample to a spin column and centrifuge. In the case of magnetic particles, pellet the magnetic particles with the aid of a magnetic field and discard the supernatant. Alternatively, separation can also be performed using spin columns.

[0244] - Add 400 μl of wash buffer (see above) to the spin column and centrifuge (Oligotex particles) or add 400 μl of wash buffer (see above) to the precipitated complexes and resuspend by vortexing before magnetic separation (magnetic particles) (alternatively, separation can also be performed using spin columns).

[0245] - Release poly(A) RNA from the washed hybrids by adding 50 μl of elution buffer (5 mM Tris-Cl, pH 7.5) (70 °C) and resuspend by pipetting up and down 3-4 times.

[0246] - Separate the poly(A)-containing eluate from the solid support by centrifugation (Oligotex particles) or magnetic separation (magnetic particles) and recover the eluate (flowthrough in the case of Oligotex particles, supernatant in the case of magnetic particles; of course, magnetic particle spin columns can also be used).

[0247] Magnetic solution

[0248] Alternatively, a further magnetic particle poly(A) nucleic acid separation protocol (magnetic protocol) may be followed. Briefly, total RNA (undiluted) is contacted with 370 μl of hybridization solution and 50 μl of magnetic particles (see above). Hybridization is carried out for 5 minutes at room temperature. After magnetic separation of the magnetic particles from the bound poly(A) nucleic acids, the particles are washed several times and the poly(A) nucleic acids are eluted with a low salt buffer.

[0249] Real-time RT-PCR analysis

[0250] RNA was measured using SYBR green reporter dye based real-time quantitative RT-PCR analysis. The following target RNAs were measured: 18S rRNA (non-poly(A) contaminant), 28S rRNA (non-poly(A) contaminant) and other rRNAs (non-poly(A) contaminant) and RPL (60S ribosomal protein L12 gene) mRNA, PPIA (peptidylprolyl isomerase A gene) mRNA, CDH2 (cadherin-2 gene) mRNA and / or GAPDH (glyceraldehyde 3-phosphate dehydrogenase gene) mRNA.

[0251] Hybridization conditions tested

[0252] 1. Comparative Example 1

[0253] Example 1 shows that the nature of the salt used for hybridization is important. Hybridization studies using hybridization solutions containing LiCl, MgCl2 and KCl showed that using these salts at different concentrations had little or no effect on the recovery of non-poly(A) (as measured by analysis of the rRNA content in the eluate). Different setups were tested:

[0254] a) Effect of reducing LiCl concentration in hybridization solution

[0255] The effect of decreasing the LiCl concentration (700 mM, 500 mM, 300 mM) in the hybridization solution on rRNA removal and poly(A) mRNA enrichment was determined by analyzing the 18s rRNA and RPL transcript levels in the eluate obtained after poly(A) enrichment. The 18s rRNA and RPL transcript levels in the initial total RNA sample were analyzed in parallel as a reference control.

[0256] 5 μg of total RNA from Jurkat cells was used as starting material and the poly(A) RNA isolation process was performed according to the magnetic protocol. The hybridization solutions tested contained Tris-buffer (pH 7.5), anionic detergents (LiDs) and different concentrations of LiCl (300 mM, 500 mM or 700 mM).

[0257] Two wash buffers were used, wash buffer 1 (150 mM LiCl, LiDs) and 2 (150 mM LiCl).

[0258] Briefly, poly(A) RNA was manually isolated as follows:

[0259] - Pipette 7.4 μl of total RNA (680 ng / μl) into an RNase-free tube. Add 370 μl of hybridization solution.

[0260] - Add 50 μl of magnetic beads with immobilized oligo d(T) capture oligonucleotide and mix the contents by gently shaking the tube.

[0261] - Incubate at room temperature for 5 minutes to allow hybridization of the poly(A) RNA to the capture oligonucleotide.

[0262] - Apply a magnetic field for 1 minute to separate the bead:poly(A)RNA complexes from the remaining sample.

[0263] - Wash ×2: For each wash step, add 300 μl of Wash Buffer 1 to the beads: poly(A) RNA pellet and resuspend by vortexing; apply a magnetic field for 1 min; discard the supernatant.

[0264] - Wash × 3: For each wash step, add 300 μl of Wash Buffer 2 to the beads:mRNA pellet and resuspend by vortexing; apply a magnetic field and incubate for 1 min; discard the supernatant.

[0265] - Add 50 μl of elution solution (low salt solution, neutral pH); incubate at 65°C for 2 minutes; apply magnetic field and incubate for 1 minute.

[0266] - Recover the supernatant containing the eluted poly(A) RNA and transfer to another RNase-free tube.

[0267] The obtained eluate was diluted 1:280 (2 μl eluate + 558 μl H2O) for real-time RT-PCR assays detecting 18S rRNA and RPL mRNA targets. The initial total RNA (680 ng / μl) was diluted 1:1838,27 to reach a concentration of 0.37 ng / μl. 5 μl of diluted poly(A) RNA eluate or diluted total RNA was used in real-time RT-PCR analysis. All pathways were analyzed in duplicate for each condition.

[0268] result

[0269] The average Ct value was calculated from two replicates of each pathway. The Ct value is the cycle threshold value ( c ycle tThe Ct value is an abbreviation for hreshold, which corresponds to the PCR cycle number at which the fluorescence of the SYBR green reporter dye first rises exponentially above the background value (threshold). The lower the Ct value, the more cDNA template there is, and therefore the more initial transcripts are present in the eluate. An increase in transcript Ct values ​​indicates that some transcripts were lost during poly(A) RNA enrichment. The results are shown in Table 1.

[0270]

[0271] All hybridization solutions tested removed 18S rRNA transcript levels, as evidenced by the significantly increased average Ct values ​​compared to the Ct values ​​obtained for the initial total RNA samples. 18S rRNA and RPL mRNA levels in the eluate were comparable in the different hybridization solutions tested. Thus, hybridization stringency was increased by reducing the LiCl salt concentration in the hybridization solution, and thus the hybridization composition had no effect on rRNA removal or mRNA enrichment.

[0272] b) Effect of reducing KCl or MgCl2 concentration in hybridization solution

[0273] In addition, the effect of reducing the concentration of KCl or MgCl2 in the hybridization solution on rRNA depletion (based on 18S rRNA) and poly(A) mRNA enrichment (based on PPIA and CDH2 transcript levels) was tested. The hybridization solution contained 20 mM Tris, 2% SDS, and 2.5 mM EDTA in addition to water and different concentrations of KCl or MgCl2 (for each salt: 1 M, 500 mM, or 100 mM); the pH was adjusted to 7.5 with HCl or NaOH. The corresponding hybridization solutions containing 1 M NaCl instead of KCl or MgCl2 were tested as reference standards.

[0274] 5 μg of total RNA purified from Jurkat cells was used as starting material. For poly(A) RNA isolation, the basic protocol under Materials and Methods above was used, using as capture oligonucleotide functionalized solid support, Oligotex suspension and spin columns to aid separation.

[0275] The obtained eluate was diluted 1:200 for real-time RT-PCR analysis. The initial total RNA (1351 ng / μl) was diluted to 3.7 μg / 46.3 μl of RNase-free water; a 1:200 dilution was obtained from this initial dilution. 5 μl of the diluted poly(A) RNA eluate or diluted total RNA was used in the real-time RT-PCR analysis.

[0276] result

[0277] The average Ct value was determined and the results are shown in Table 2.

[0278]

[0279] All hybridization solutions tested depleted transcript levels of 18S rRNA, as evidenced by the increased mean Ct values ​​compared to those obtained for the initial total RNA sample. Reductions in the KCl salt concentration in the hybridization solution resulted in inconclusive results for potential improvements in rRNA removal. Reducing the KCl salt from 1 M to 500 mM in the hybridization solution resulted in lower rRNA removal efficiency compared to the 1 M KCl approach, while the hybridization solution containing 100 mM KCl removed more rRNA than the hybridization buffer containing 1 M KCl. Reductions in the MgCl2 concentration in the hybridization solution were negatively correlated with 18S rRNA removal. 1 M MgCl2 achieved the best rRNA removal in the hybridization solution, but was associated with a significant loss of target mRNA. The lowest concentration of MgCl2 tested (1 00 mM) showed only good rRNA removal and mRNA enrichment efficiency similar to that of the hybridization solution containing 1 M NaCl.

[0280] 2. Comparative Example 2

[0281] The effect of reducing the concentration of NaCl in the hybridization solution on rRNA depletion (based on 18S and 28S rRNA) and poly(A) mRNA enrichment (based on GPDH and PPIA mRNA) was analyzed. The hybridization solution contained 20 mM Tris (pH 7.5), 2% SDS, and 2.5 mM EDTA (pH 8) in addition to water and different concentrations of NaCl (1 M, 400 mM, 300 mM, 200 mM, 100 mM, or 50 mM).

[0282] Poly(A) RNA was isolated following the basic protocol described above under Materials and Methods, using capture oligonucleotide functionalized solid supports, Oligotex suspensions and spin columns to aid in the separation. 5 μg of total RNA purified from Jurkat cells was used as starting material.

[0283] The obtained eluate was diluted 1:1000 for real-time PCR assays of rRNA and mRNA targets. The initial total RNA (1.27 μg / μl) was diluted by adding 3.94 μl of RNA to 46.06 μl of RNase-free water. 5 μl of diluted poly(A) RNA eluate or diluted total RNA was used for real-time RT-PCR analysis.

[0284] result

[0285] The average Ct value was determined and the results are shown in Table 3. Figure 1The ΔCt values ​​of the conditions tested are shown (ΔCt values ​​were calculated by subtracting the average Ct values ​​measured from the initial total RNA samples from the average Ct values ​​determined after poly(A) RNA enrichment). All hybridization solutions tested removed 18S rRNA during poly(A) enrichment, as evidenced by the significantly increased average Ct values ​​compared to the average Ct values ​​obtained from the initial total RNA samples.

[0286]

[0287] All tested hybridization solutions removed 18S rRNA transcript levels, as evidenced by the significantly increased mean Ct values ​​compared to the Ct values ​​obtained for the initial total RNA sample. It can be observed that there is a substantial reduction in rRNA levels in a concentration-dependent manner. The lower the concentration of NaCl in the hybridization solution, the less rRNA contamination there is in the eluate, and the more efficient the rRNA removal. The maximum rRNA reduction was achieved when the NaCl concentration in the hybridization solution was below 200 mM, as evidenced by the significantly increased Ct values. However, the reduction in NaCl concentration in the hybridization solution had a less dramatic effect on mRNA recovery. Therefore, relatively speaking, due to more stringent hybridization conditions, more rRNA is removed, while less target mRNA is lost. It can be seen that reducing the concentration of NaCl in the hybridization solution has a dramatic effect on the recovery of 18S and 28S rRNA, while having only some effect on the recovery of mRNA. This trend is also reflected by Figure 1 However, even though the recovery of non-poly(A) RNA was greatly affected, lowering the NaCl concentration to 200 mM still resulted in a loss of mRNA in these hybridization conditions, as evidenced by the increase in the Ct values ​​for the mRNA target.

[0288] In addition, in additional experiments, the amount of SDS was varied (0%, 0.2% and 2%). This variation had no effect on hybridization (as rRNA removal and mRNA recovery remained approximately the same (data not shown)).

[0289] 3. Example 3

[0290] In Example 3, the effect of low ionic strength NaCl salt concentration (150 mM) in combination with different quaternary ammonium salts in the hybridization solution on rRNA removal and poly(A) mRNA enrichment was analyzed. The test hybridization solution had the following composition: 20 mM Tris, 150 mM NaCl, 0.2% SDS, EDTA, water and 1 M of the test quaternary ammonium salt. As quaternary ammonium salts, tetraalkylammonium salts of tetramethylammonium chloride (TMAC), tetramethylammonium nitrate (TMA nitrate), tetramethylammonium bromide (TMA bromide) or tetraethylammonium bromide (TEA bromide) were tested. The poly(A) separation procedure was performed using the basic protocol under the above materials and methods, using Seradyn magnetic beads as a solid support functionalized with capture oligonucleotides.

[0291] Total RNA (0.5 μg and 2 μg) from Jurkart cells was used as starting material. rRNA depletion was tested based on 18S rRNA and 28S rRNA levels, and poly(A) RNA enrichment was tested based on PPIA and GAPDH transcripts.

[0292] The obtained eluate was diluted 1:200 for real-time RT-PCR testing of rRNA and mRNA targets. 6.37 μl was added to 43.63 μl of RNase-free water to dilute the initial total RNA (785.5 ng / μl). A 1:200 dilution was obtained from this initial dilution. 5 μl of the diluted poly(A) RNA eluate or diluted total RNA was used in the real-time RT-PCR analysis. Using 0.5 μg or 2 μg of total RNA as starting material, the average Ct values ​​for 18S rRNA and 28S rRNA as well as GAPDH mRNA and PPIA mRNA for each condition were calculated from two parallel experiments.

[0293] result

[0294] Figure 2a ) to d) show the ΔCt values ​​obtained with the tested conditions (calculated as described above). It can be seen that the effects on rRNA removal and mRNA recovery were comparable when either 0.5 μg or 2.0 μg of total RNA was used as starting material for poly(A) RNA isolation. Compared to the 1 M NaCl reference hybridization solution, all tested hybridization solutions containing 150 mM NaCl and quaternary ammonium salts resulted in 18S rRNA ( Figure 2a ) and 28S rRNA ( Figure 2b ) removal rate is significantly higher. In addition, Figure 2c) and d) As can be seen, the hybridization solution using 150 mM NaCl in combination with tetraalkylammonium salts showed significantly less unwanted loss of poly(A) RNA compared to the hybridization solution containing 150 mM NaCl, as determined based on the target mRNAs PPIA and GAPDH. Thus, mRNA loss can be significantly reduced compared to the hybridization solution without tetraalkylammonium salts.

[0295] Therefore, the combination of low concentrations of NaCl in the hybridization solution (and its hybridization composition) and the addition of quaternary ammonium salts forms a favorable hybridization condition, wherein the unwanted rRNA is effectively removed, and the desired poly (A) RNA is effectively hybridized to the capture oligonucleotide and enriched thereby. Therefore, the hybridization conditions taught by the present invention (wherein a relatively low concentration of sodium salt such as sodium chloride is used in combination with a quaternary ammonium salt) provide a favorable balance between the removal of non-poly (A) and the recovery of poly (A) RNA.

[0296] 4. Example 4

[0297] Here, different concentrations of TMA bromide in the hybridization solution were used and the effect on rRNA removal and poly(A) mRNA enrichment was analyzed. The hybridization solutions tested had the following composition: 20 mM Tris, 150 mM NaCl, 0.2% SDS and EDTA, water and 0.5 M, 1 M, 1.5 M or 2 M TMAB. As a reference, the corresponding hybridization solutions containing 1 M or 150 mM NaCl but without quaternary ammonium salts were tested in parallel.

[0298] The isolation procedure of poly(A) RNA was performed using the basic protocol under the above materials and methods, using Seradyn magnetic beads as a solid support functionalized with capture oligonucleotides. 2 μg of total RNA isolated from Jurkart cells was used as starting material. The removal of rRNA was tested based on 18S rRNA and 28S rRNA levels, and the enrichment of poly(A) RNA was tested based on PPIA and GAPDH mRNA.

[0299] For real-time RT-PCR assays against rRNA and mRNA targets, the obtained eluate was diluted 1:200. The initial total RNA (0.97 μg / μl) was diluted by adding 2.06 μl to 47.95 μl of RNase-free water. A 1:200 dilution was obtained from this initial dilution. 5 μl of the diluted poly(A) RNA eluate or diluted total RNA was used in the real-time RT-PCR analysis.

[0300] result

[0301] Figure 3a ) to 3d) show the results:

[0302] Figure 3a ) shows the ΔCt values ​​obtained for 18S and 28S rRNA. It can be seen that the rRNA removal results are improved compared to the 1 M NaCl reference hybridization solution. Figure 3b ) shows the ΔCt values ​​obtained for GAPDH and PPIA target mRNAs. It can be seen that the hybridization solutions of the present invention obtained improved results compared to the 150 mM and 1 M NaCl reference hybridization solutions under all tested conditions.

[0303] Figure 3c ) shows the ΔΔCt value of GAPDH, Figure 3d ) ΔΔCt value of PPIA. The ΔΔCt value was calculated from the ΔCt value (the average CT value under each condition normalized to the corresponding initial total RNA control) according to the following formula: the ΔCt value of 18SrRNA or 28S rRNA under each condition minus the ΔCt value of GAPDH mRNA level or PPIA mRNA level under each condition. A higher ΔΔCt value is advantageous because it shows a high rRNA removal rate. It can be seen that the results are improved compared to the hybridization solution containing 1M NaCl. In addition, the hybridization solution of the present invention obtained similar or better ΔΔCt values ​​compared to the hybridization solution containing 150mM NaCl. Therefore, the ΔΔCt results are maintained or even improved compared to the hybridization solution containing 150mM salt, and importantly, the recovery of mRNA is significantly improved using the hybridization solution of the present invention, as shown Figure 3a ) and b).

[0304] Thus, Example 4 clearly demonstrates the benefits of the hybridization solutions and hybridization compositions of the present invention and shows that tetraalkylammonium salts can be used at different concentrations. These results were also confirmed in other experiments, where corresponding hybridization solutions containing 0.2 MTMAB were tested. Thus, quaternary ammonium salts work at high and low concentrations.

[0305] 5. Example 5

[0306] The isolation procedure of poly(A)RNA was performed using the basic protocol under Materials and Methods above, using Seradyn magnetic beads as solid support functionalized with capture oligonucleotides. The hybridization solution shown contained 20 mM Tris, 150 mM NaCl, 0.2% SDS water and 1 M TMAB. 5 μg of total RNA isolated from Jurkart cells was used as starting material. The residual amount of different rRNAs (5S rRNA, 5.8S rRNA, 12s rRNA, 16SrRNA, 18S rRNA and 28S rRNA) was determined in the isolated poly(A)RNA eluate based on real-time RT-PCR analysis. Seven samples were evaluated. The resulting eluate was diluted 1:1000. The initial total RNA was diluted to 5.1 μg / 50 μl. A 1:1000 dilution was obtained from this initial dilution. An additional 1:10 dilution was then prepared. 5 μl of the diluted poly(A)RNA eluate or the diluted total RNA was used in the real-time RT-PCR analysis.

[0307] result

[0308]

[0309] It can be seen that different types of rRNA were effectively removed, usually ≥99% or even 100%, which shows that the hybridization conditions of the present invention can achieve excellent removal effects.

[0310] 6. Comparative Example 6

[0311] In Example 6, the effect of adding different substances (DMSO, formamide, TMAC, betaine) to a hybridization solution containing 500 mM LiCl was tested. 5 μg of total RNA isolated from Jurkart cells was used as starting material for the enrichment of poly(A) nucleic acids using the magnetic scheme described above. 4.20 μl of total RNA, 370 μl of hybridization solution and 50 μl of magnetic particles functionalized with capture oligonucleotides were mixed to prepare a hybridization composition.

[0312] Reference LiCl buffer: (Tris pH: 7.5, 500 mM LiCl, 1% anionic detergent)

[0313] LiCl-DMSO: LiCl buffer + DMSO as reference composition (final: 5%)

[0314] LiCl-formamide: LiCl buffer + formamide as reference composition (final: 2%)

[0315] LiCl-TMAC: LiCl buffer + TMAC as reference composition (final: 100 nM)

[0316] LiCl-Betaine: LiCl buffer + Betaine as reference composition (final: 1M)

[0317] result

[0318] The results of real-time quantitative RT-PCR analysis are shown in Table 5. The ΔCt values ​​of 18S and 28S rRNA were measured to analyze the depletion of non-poly(A), and the ΔCt values ​​of GAPDH and CDH2 were measured to analyze the enrichment of mRNA.

[0319]

[0320] In addition, the reference hybridization buffer (see above) was supplemented with different concentrations of TMAC (see Table 6 below) to analyze whether higher concentrations of TMAC would improve the results. However, the combination with LiCl did not see any effect on mRNA recovery or rRNA removal. This example was repeated at different hybridization temperatures (40°C, 50°C and 60°C) and had no effect.

[0321]

[0322] Taken together with the previous experiments, these results demonstrate that the combination of NaCl concentration and quaternary ammonium salts in the hybridization solution can achieve significant removal of non-poly(A) RNA after enrichment of poly(A) RNA, while retaining mRNA bound to the oligonucleotide capture probes, thereby ensuring efficient and selective recovery of poly(A) RNA.

Claims

1. A method for separating a poly(A) nucleic acid having a single-stranded poly(A) extension from a sample containing nucleic acid, comprising: (a) providing a hybridization composition comprising: i) samples containing nucleic acids; ii) a hybridization solution comprising: aa. Sodium chloride; bb. a tetraalkylammonium salt selected from the group consisting of tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB); wherein the components of the hybridization solution are added to the sample as a single solution or are added to the sample separately in any order; iii) a capture probe capable of hybridizing to a poly(A) stretch of a poly(A) nucleic acid; and incubating the hybridization composition under conditions such that a nucleic acid hybrid is formed between the poly(A) nucleic acid and the capture probe; (b) separating the formed hybrid from the remaining sample, in, The hybridization composition comprises a hybridization solution sodium chloride having a concentration of 25 mM to 175 mM and a hybridization solution tetraalkylammonium salt having a concentration of 0.1 M to 1.25 M; and / or The hybridization solution contains sodium chloride at a concentration of 50 mM to 350 mM and a tetraalkylammonium salt at a concentration of 0.2 M to 2.5 M.

2. The method of claim 1, wherein the tetraalkylammonium salt is tetramethylammonium bromide.

3. The method of claim 1, wherein the hybridization composition is incubated under conditions such that nucleic acid hybrids are formed between the poly(A) nucleic acid and the capture probe. The method of claim 1 , wherein the poly(A) nucleic acid is poly(A) RNA.

5. The method of claim 1, wherein the nucleic acid-containing sample is total RNA.

6. The method of claim 1 for isolating poly(A) RNA from a total RNA sample, comprising: (a) providing a hybridization composition comprising: i) samples containing nucleic acids; ii) a hybridization solution comprising: aa. Sodium chloride, concentration 75mM-250mM; bb. A tetraalkylammonium salt selected from the group consisting of tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB), at a concentration of 0.2M-2.5M; wherein the components of the hybridization solution are added to the sample as a single solution or separately in any order; iii) a capture probe capable of hybridizing to a poly(A) stretch of a poly(A) nucleic acid, wherein the capture probe is immobilized on a solid support; The hybridization composition comprises a hybridization solution sodium chloride at a concentration of 37.5 mM to 125 mM and a tetraalkylammonium salt at a concentration of 0.125 M to 1.25 M, and incubating the hybridization composition under conditions such that a nucleic acid hybrid is formed between the poly(A) nucleic acid and the capture probe; (b) Separating the formed hybrids from the remaining sample.

7. The method of claim 1, wherein the capture probe is a capture oligonucleotide comprising a single-stranded sequence complementary to a poly(A) stretch of a poly(A) nucleic acid.

8. The method of claim 1, wherein the capture probe is an oligo(T)- or oligo(U)-containing oligonucleotide.

9. The method of claim 1, wherein the capture probe is bound to a solid support.

10. The method of claim 9, wherein the solid support is provided by particles. The method of claim 10 , wherein the particles are magnetic particles.

12. The method of claim 1, wherein the hybridization composition is incubated at an elevated temperature of 60°C or higher and then incubated at a temperature of 40°C or lower to allow the poly(A) RNA to hybridize to the capture probe.

13. The method of claim 1, wherein the method comprises the following additional steps: (c) optionally washing and (d) releasing poly(A) nucleic acid from the washed hybrid.

14. The method of claim 1, wherein the hybridization composition contains a detergent and / or a chelating agent.

15. The method of claim 1, wherein a single poly(A) nucleic acid isolation cycle is performed to isolate poly(A) nucleic acid.

16. The method of claim 15, wherein the isolated poly(A) nucleic acid is poly(A) RNA, and wherein the method comprises sequencing the isolated poly(A) RNA.

17. The method of claim 16, wherein the sequencing is performed by next generation sequencing.

18. A method for sequencing poly(A) nucleic acid, comprising: (a) isolating poly(A) nucleic acid from a sample containing nucleic acid using the method according to any one of claims 1 to 17; (b) Sequencing the isolated poly(A) nucleic acid molecules. The method of claim 18 , wherein the poly(A) nucleic acid is poly(A) RNA.

20. A water-soluble hybridization solution suitable for hybridizing a poly(A) nucleic acid with a capture probe, wherein the capture probe is capable of hybridizing to a poly(A) stretch of the poly(A) nucleic acid, comprising: aa. Sodium chloride, concentration 50mM-350mM; bb. A tetraalkylammonium salt selected from the group consisting of tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB) at a concentration of 0.2M to 2.5M.

21. The aqueous hybridization solution of claim 20, wherein the hybridization solution contains sodium chloride at a concentration of 75 mM to 250 mM and a tetraalkylammonium salt at a concentration of 0.25 M to 2.5 M, wherein the tetraalkylammonium salt is selected from the group consisting of tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB).

22. A kit for isolating poly(A) nucleic acid from a sample containing nucleic acid, comprising: (a) the hybridization solution according to any one of claims 20 to 21; (b) Capture probe capable of hybridizing to a poly(A) stretch of poly(A) nucleic acid.

23. A method for isolating a poly(A) nucleic acid having a single-stranded poly(A) stretch from a sample containing nucleic acid, comprising: (a) hybridizing a poly(A) nucleic acid with a capture probe to form a nucleic acid-hybrid between the poly(A) nucleic acid and the capture probe, wherein the capture probe is capable of hybridizing to a poly(A) stretch of the poly(A) nucleic acid; (b) separating the formed hybrids from the remaining sample; (c) washing the separated hybrids using the hybridization solution of any one of claims 20 to 21, wherein the components of the hybridization solution are added to the hybrids as a single solution or are added to the hybrids separately in any order to form the hybridization solution for washing; (d) releasing the poly(A) nucleic acid from the washed hybrid.

24. The method of claim 23, wherein the addition of the hybridization solution and optionally the dilution solution results in the creation of a wash composition, wherein the wash composition has one or more of the following characteristics: a) the washing composition contains hybridization solution sodium chloride at a concentration of ≤250 mM; and / or b) The washing composition contains a hybridization solution tetraalkylammonium salt at a concentration of ≤1.5M.

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