Method for preparing standardized nucleic acid sample, kit and device therefor

Abstract

Methods and devices for preparing treated RNA and DNA samples are provided. A first nucleic acid sample is used to generate a set of probes based on intrinsic sequence abundance in the sample. The abundant sequences will produce more probes. When a second nucleic acid sample is applied to the probe, more abundant sequences will bind to the probe so that these sequences can be isolated from the sample. Methods of extracting RNA and detecting target sequences using the probes are also provided. A device for microfluidic processing of RNA in a flow path is also claimed. A method for treating nucleic acids, wherein a surface comprising a probe of more than 100 nucleotides in length is used.

Description

Technical Field

[0001] The present invention relates to methods and devices for preparing processed RNA and DNA samples. The present invention also relates to detecting target nucleic acids. Methods for analyzing biological samples using the processing and detection methods are also provided. Background Art

[0002] RNA sequencing has become a powerful tool for understanding biology (Stark, R., Grzelak, M. & Hadfield, J. RNA sequencing: the teenage years. Nat. Rev. Genet. 20, 631-656 (2019)). Its applications range from drug development to improved agriculture. RNA sequencing is often used to identify differences between biological samples. These samples can be samples from infected and control animals for studying disease resistance, or samples from the same sample type over a period of time for understanding growth and development. The main results produced by RNA sequencing are the discovery of all genes and isoforms expressed in the sample and the quantification of expression. Most cells and tissues share many of the same highly expressed genes, which are often referred to as housekeeping genes. These genes are generally responsible for basic cellular functions and therefore do not provide cell-specific features. Since these housekeeping genes usually constitute the majority of RNA in the sample, RNA sequencing data is usually dominated by sequencing reads of these uninformative RNAs. This phenomenon has two major negative effects on generating good results from RNA sequencing projects; first, genes and isoforms specific for the disorder in question are difficult to detect, and second, the data generated are largely redundant.

[0003] The first major negative effect has two consequences. First, the amount of sequencing required to detect the target gene must be large enough to handle the sampling inefficiency caused by the low relative abundance of the target gene. The second is that, in some cases, low-abundance target genes may not be identified at all. This can be demonstrated by the ongoing efforts to annotate the human genome. Even after thousands of sequencing projects, the complete human transcriptome remains elusive, with new isoforms and genes being reported regularly. Since the complexity of the eukaryotic transcriptome comes from the selective splicing that produces combinatorial arrangements, finding new RNAs will likely be a continuous effort.

[0004] These two consequences ultimately hinder scientific progress by limiting the ability of researchers to produce the desired results from their sequencing experiments. These consequences also make it infeasible to apply RNA sequencing to a wider range of uses. For example, when used for diagnosis and treatment tracking, the amount of sequencing required is prohibitive in both time and cost.

[0005] The second major negative effect (generating redundant data) also has two major consequences. The first is that more data requires more processing time, which increases the overall cost and time of RNA sequencing experiments. These costs include both the energy required for additional calculations and the working time of the bioinformatician responsible for processing the data. The second consequence is that redundant data leads to the need for more storage. As sequencing becomes more common, data storage has become an important issue. In order for RNA sequencing technology to take on more roles, more efficient data generation is needed to reduce storage requirements.

[0006] In order to solve the problem that high-abundance housekeeping genes reduce the sampling efficiency of target genes, complementary DNA (cDNA) standardization was developed (Alex S. Shcheglov, Pavel A. Zhulidov, Ekaterina A. Bogdanova, DAS Normalization of cDNA Libraries, Nucleic Acids Hybrid. CHAPTER 5, (2014)). Since RNA sequencing usually relies on the conversion of RNA to double-stranded cDNA, cDNA standardization uses the biochemical properties of cDNA to produce a uniform distribution of unique genes and isoforms in the cDNA library. In theory, if all unique RNA sequences are expressed in the same relative abundance, the maximum non-targeted sampling efficiency is generated. Therefore, the purpose of standardization is to redistribute the cDNA library to meet the standard as closely as possible.

[0007] Two forms of full-length cDNA standardization have been developed previously: double-stranded specific nuclease (DuplexSpecific Nuclease, DSN) method (Zhulidov, PA et al. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Res. 32, e37 (2004)) and hydroxyapatite column method (Andrews-Pfannkoch, C., Fadrosh, DW, Thorpe, J. & Williamson, SJ Hydroxyapatite-mediated separation of double-stranded DNA, single-strandedDNA, and RNAgenomes from natural viral assemblages. Appl. Environ. Microbiol. 76, 5039–5045 (2010)). Both methods rely on denaturation and re-hybridization of cDNA chains. Since single-stranded cDNA moves everywhere in the solution, sequences with higher abundance have a greater probability of finding complementary sequences that match them for re-hybridization. Therefore, when rehybridization reaches its limit, the remaining single-stranded cDNA represents the normalized sequence library.

[0008] The difference between these two methods lies in the method used to separate the single-stranded cDNA library from the rehybridized double-stranded cDNA molecules.

[0009] In the DSN method, an enzyme that specifically cuts double-stranded DNA is used to break down all double-stranded cDNA in a solution. The solution is then purified and cDNA sequences that exceed a certain length are size-selected. These sequences are then amplified using the polymerase chain reaction (PCR).

[0010] In the column method, the denatured and rehybridized cDNA library is passed through a heated column filled with hydroxyapatite particles. Hydroxyapatite preferentially binds to larger DNA molecules. The size of the bound DNA is controlled by the concentration of the phosphate buffer in which the cDNA library is dissolved. Therefore, the concentration of the phosphate buffer must be adjusted specifically for cDNA molecules within a certain sequence length range. The cDNA is eluted through the column using an increased concentration of phosphate buffer to extract the DNA molecules of increased size. Since single-stranded cDNA is roughly half the size of rehybridized cDNA, if the average cDNA sequence length is known, the elution of the single-stranded fraction can be managed. The elution that occurs is intended to enrich the single-stranded cDNA, which is then amplified using PCR.

[0011] Since the DSN method uses an enzyme that cuts all double-stranded cDNAs, it can theoretically deplete low-abundance sequences with segments that match high-abundance sequences. This effect can also increase the possibility of forming PCR chimeras. When incomplete single-stranded cDNA sequences act as primers for other sequences, thereby combining sequences in a way that does not exist in nature, PCR chimeras are formed. PCR chimeras represent false positives of new isoforms and are extremely challenging to distinguish from true alternative isoforms. Verifying PCR chimeras usually requires in-depth biochemical assays. Both the depletion of low-abundance sequences and the potential increase of PCR chimeras make the DSN method unsuitable for many RNA sequencing applications.

[0012] Since the column method only allows separation of high and low abundance fractions within a narrow size range, it has a significant bias towards longer cDNA sequences. The result is a loss of representation of longer RNA sequences. This effect makes it unsuitable for many RNA sequencing applications.

[0013] Therefore, it is with these problems in mind that the present invention has been designed. Summary of the invention

[0014] RNA or cDNA samples are usually dominated by sequences from highly expressed genes, which can have a negative impact on the analysis of samples. The inventors have developed methods and devices for preparing processed nucleic acid samples with more uniform sequence distribution. The first nucleic acid sample is used to produce probe groups based on the intrinsic sequence abundance in the sample. Abundant sequences will produce more probes. When the second nucleic acid sample is applied to the probe, more abundant sequences will be combined with the probe, making it possible to separate these sequences from the sample. In this way, the present invention can standardize full-length RNA and cDNA. This technology is also suitable for extracting the method for RNA and the detection of specific target sequences. It is also useful in the method and diagnostic method of analyzing biological samples according to RNA and DNA processing of the present invention.

[0015] It should be remembered that multiple aspects have been designed to be advantageously combined, and all such combinations are considered to be within the scope of the present invention. It should also be understood that, where appropriate, the selections described with respect to one area of ​​improvement will be applied to other areas after necessary modifications; e.g., sample type, nucleic acid type, etc.

[0016] In a first aspect, the present invention provides a method for processing nucleic acids, the method comprising:

[0017] (i) contacting a nucleic acid sample (e.g., an RNA sample) with an oligonucleotide array (e.g., a DNA, optionally a cDNA array), wherein one or more nucleic acid molecules from the nucleic acid sample anneal to one or more oligonucleotides in the oligonucleotide array; and

[0018] (ii) extracting the unannealed nucleic acid molecules, thereby producing treated nucleic acids.

[0019] In some embodiments, the array is produced by a method comprising:

[0020] (i) contacting an RNA sample with a surface, wherein the surface comprises two or more oligo-dT molecules, and wherein two or more RNA molecules from the RNA sample anneal to the oligo-dT molecules;

[0021] (ii) using the annealed RNA molecules as templates, extending two or more of the oligo dT molecules by reverse transcription to generate a DNA array comprising two or more cDNA molecules;

[0022] (iii) dissociating the annealed RNA molecules from the cDNA molecules;

[0023] (iv) Removal of RNA sample from the surface.

[0024] Thus, in these embodiments, the array comprises two or more oligonucleotides having a sequence comprising oligo dT followed by a cDNA sequence.

[0025] In other embodiments, the array is produced by a method comprising:

[0026] (i) denaturing a cDNA sample comprising double-stranded cDNA molecules to generate single-stranded cDNA molecules;

[0027] (ii) contacting the cDNA sample with a surface, wherein the surface comprises two or more oligo-dT molecules, and wherein two or more cDNA molecules from the cDNA sample anneal to the oligo-dT molecules;

[0028] (iii) extending two or more of the oligo dT molecules using the annealed cDNA molecules as templates to generate a DNA array comprising the two or more DNA molecules;

[0029] (iv) dissociating the annealed cDNA molecules from the DNA molecules;

[0030] (v) Removing the cDNA sample from the surface.

[0031] Thus, in these embodiments, the array comprises two or more oligonucleotides having a sequence comprising oligo dT followed by a DNA sequence.

[0032] According to a related aspect of the present invention, there is provided a method for processing nucleic acid, the method comprising:

[0033] (i) contacting a first nucleic acid sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, and wherein two or more nucleic acid molecules from the first nucleic acid sample anneal to the oligonucleotides in the oligonucleotide array;

[0034] (ii) extending two or more of the oligonucleotides using the annealed nucleic acid molecules as templates to generate a DNA array comprising the two or more DNA molecules;

[0035] (iii) dissociating the annealed nucleic acid molecules from the DNA molecules;

[0036] (iv) removing the first nucleic acid sample from the surface;

[0037] (v) contacting a second nucleic acid sample with the DNA array, wherein one or more nucleic acid molecules from the second nucleic acid sample anneal to the DNA molecules; and

[0038] Unannealed nucleic acid molecules are extracted, thereby producing treated nucleic acids.

[0039] According to the present invention, nucleic acid is not restrictive. Any suitable nucleic acid molecule can be processed using the device, test kit and method of the present invention. Nucleic acid can be double-stranded or single-stranded. According to all aspects of the present invention, when the nucleic acid molecule is double-stranded, first the double-stranded nucleic acid molecule is denatured to produce a single-stranded nucleic acid molecule.

[0040] Nucleic acid can be DNA. DNA can be genomic DNA, mitochondrial DNA, cDNA, etc. Preferably cDNA. DNA can be purified from any suitable sample. Sample types include blood samples (especially blood samples from plasma and serum); other body fluids, such as saliva, urine or lymph. Other sample types include solid tissues, including frozen tissues or formalin fixed paraffin embedded (FFPE) materials. DNA molecules can be double-stranded DNA (double-stranded DNA, dsDNA) molecules. In some alternative embodiments, DNA molecules are single-stranded DNA (ssDNA) molecules. In some embodiments, ssDNA has been denatured in situ in the original sample. For example, ssDNA can be purified from FFPE materials. In other embodiments, nucleic acid samples can include both ssDNA molecules and dsDNA molecules. For example, in the case of DNA purified from FFPE materials, DNA can include both ssDNA and dsDNA. DNA can be present in cells in the sample, or derived from cells in the sample. Alternatively, the DNA may be circulating or "cell-free" DNA (cfDNA). Such DNA may be obtained from a range of body fluids, including blood samples (particularly blood samples from plasma and serum); other body fluids, such as saliva, urine or lymph.

[0041] The nucleic acid may also be RNA. RNA may be obtained from the same sample types as DNA, as discussed above. RNA may be messenger RNA (mRNA), microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (lncRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), small rDNA-derived RNA (srRNA), viral RNA, etc. mRNA is preferred.

[0042] Therefore, the present invention provides a method for processing RNA, the method comprising:

[0043] (i) contacting a first RNA sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, and wherein two or more RNA molecules from the first RNA sample anneal to the oligonucleotides in the oligonucleotide array;

[0044] (ii) extending two or more of the oligonucleotides by reverse transcription using the annealed RNA molecules as templates to generate a DNA array comprising two or more cDNA molecules;

[0045] (iii) dissociating the annealed RNA molecules from the cDNA molecules;

[0046] (iv) removing the first RNA sample from the surface;

[0047] (v) contacting a second RNA sample with a DNA array, wherein one or more RNA molecules from the second RNA sample anneal to the cDNA molecules; and

[0048] (vi) extracting the unannealed RNA molecules, thereby producing processed RNA.

[0049] According to another aspect of the present invention, there is provided a method for processing cDNA, the method comprising:

[0050] (i) denaturing a first cDNA sample comprising double-stranded cDNA molecules to generate single-stranded cDNA molecules;

[0051] (ii) contacting the first cDNA sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, and wherein the two or more cDNA molecules from the first cDNA sample anneal to the oligonucleotides in the oligonucleotide array;

[0052] (iii) extending two or more of the oligonucleotides using the annealed cDNA molecules as templates to generate a DNA array comprising the two or more DNA molecules;

[0053] (iv) dissociating the annealed cDNA molecules from the DNA molecules of the DNA array;

[0054] (v) removing the first cDNA sample from the surface;

[0055] (vi) denaturing the second cDNA sample comprising double-stranded cDNA molecules to generate single-stranded cDNA molecules;

[0056] (vii) contacting the second cDNA sample with the DNA array, wherein one or more cDNA molecules from the second cDNA sample anneal to the DNA molecules;

[0057] (viii) extracting the unannealed cDNA molecules to generate processed cDNA.

[0058] "Array" means a collection or arrangement of oligonucleotide (DNA, optionally cDNA) molecules connected or attached to a (solid) surface. A variety of methods for connecting oligonucleotides to surfaces are available (e.g., amine-modified oligonucleotides covalently linked to activated carboxylate groups or succinimidyl esters, thiol-modified oligonucleotides covalently linked by alkylating agents such as iodoacetamide or maleimide, digoxin NHS esters, cholesterol-TEG, biotin-modified oligonucleotides captured by fixed streptavidin), and are well known to technicians. Connections can be covalent or non-covalent. Connections can be direct or indirect. DNA arrays can be cDNA arrays.

[0059] In some embodiments, the method for treating RNA reduces the variability of RNA levels (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%). The method for treating RNA can achieve a more uniform distribution of RNA sequences. The difference in abundance between the most abundant RNA and the least abundant RNA can be reduced (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%). In certain embodiments, the method for treating RNA reduces the number of molecules (copy number) of the most abundant RNA molecules (1, 10, 100, 1000 or 10000) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%). In some specific embodiments, the number of molecules (copy number) of the most abundant RNA molecule in the (second) RNA sample is reduced by at least 50% in the treated RNA. In other embodiments, the relative abundance of the (1, 10, 100, 1000 or 10000) least abundant RNA molecules is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. Therefore, the method for treating RNA can be a method for standardizing RNA.

[0060] In some specific embodiments, the method for processing cDNA reduces the variability of cDNA levels (e.g., reduces by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%). The method for processing cDNA can achieve a more uniform distribution of cDNA sequences. The difference in abundance between the most abundant cDNA and the least abundant cDNA can be reduced (e.g., reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%). In certain embodiments, the method for processing cDNA reduces the number of molecules (copy number) of the most abundant cDNA molecules (1, 10, 100, 1000 or 10000) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%). In some specific embodiments, the number of molecules (copy number) of the most abundant cDNA molecule in the (second) cDNA sample is reduced by at least 50% in the processed cDNA. In other embodiments, the relative abundance of the least abundant cDNA molecule (1, 10, 100, 1000 or 10000) is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. Therefore, the method for processing cDNA can be a method for standardizing cDNA.

[0061] In some embodiments, the method for treating nucleic acids reduces the variability of nucleic acid levels (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). The method for treating nucleic acids can achieve a more uniform distribution of nucleic acid sequences. The difference in abundance between the most abundant nucleic acid and the least abundant nucleic acid can be reduced (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). In certain embodiments, the method for treating nucleic acids reduces the number of molecules (copy number) of the most abundant nucleic acid molecules (1, 10, 100, 1000 or 10,000) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some specific embodiments, the number of molecules (copy number) of the most abundant nucleic acid molecule in the (second) nucleic acid sample is reduced by at least 50% in the treated nucleic acid. In other embodiments, the relative abundance of the least abundant nucleic acid molecule (1, 10, 100, 1000 or 10000) is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. Therefore, the method for treating nucleic acids can be a method for standardizing nucleic acids.

[0062] In theory, maximum nontargeted sampling efficiency occurs if all unique nucleic acid sequences are represented at the same relative abundance. Therefore, the goal of normalization is to redistribute nucleic acid samples to meet this criterion as closely as possible.

[0063] In certain embodiments, the processed RNA, DNA or nucleic acid is the RNA, DNA or nucleic acid that is easier to analyze. It can be sequenced more effectively because the relative representativeness of less abundant sequences is improved. Therefore, the processed RNA, DNA or nucleic acid can be a standardized RNA, DNA or nucleic acid, respectively.

[0064] In some embodiments, the treated RNA comprises RNA sequences having substantially the same levels. For example, wherein the sequence levels of the treated RNA vary by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. The treated RNA can be a treated RNA sample in which at least a portion of the most abundant sequences (1, 10, 100, 1000, or 10,000) in the second RNA sample has been removed.

[0065] In some specific embodiments, the processed cDNA comprises cDNA sequences having substantially the same level. For example, wherein the sequence levels of the processed cDNA vary by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. The processed cDNA can be a processed cDNA sample in which at least a portion of the most abundant sequences (1, 10, 100, 1000, or 10,000) in the second cDNA sample has been removed.

[0066] In some embodiments, the treated nucleic acids comprise nucleic acid sequences having substantially the same levels. For example, wherein the sequence levels of the treated nucleic acids vary by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. The treated nucleic acids can be treated nucleic acid samples in which at least a portion of the most abundant sequences (1, 10, 100, 1000, or 10,000) in the second nucleic acid sample has been removed.

[0067] According to a related aspect of the present invention, there is provided a method for preparing standardized RNA, the method comprising:

[0068] (i) providing a first RNA sample;

[0069] (ii) contacting the first RNA sample with a surface, wherein the surface comprises one or more oligonucleotides, and wherein one or more RNA molecules from the first RNA sample anneal to the one or more oligonucleotides;

[0070] (iii) reverse transcribing one or more RNA molecules using one or more of the oligonucleotides as primers to produce one or more cDNA molecules;

[0071] (iv) dissociating the one or more RNA molecules from the one or more cDNA molecules;

[0072] (v) removing the first RNA sample;

[0073] (vi) providing a second RNA sample;

[0074] (vii) contacting a second RNA sample with a surface, wherein one or more RNA molecules from the second RNA sample anneal to the one or more cDNA molecules; and

[0075] (viii) Extracting unannealed RNA molecules for use as standard RNA samples.

[0076] In another aspect, the present invention provides a method for preparing a normalized cDNA, comprising:

[0077] (i) providing a first cDNA sample comprising double-stranded cDNA molecules;

[0078] (ii) denaturing the first cDNA sample to generate single-stranded cDNA molecules;

[0079] (iii) contacting the first cDNA sample with a surface, wherein the surface comprises one or more oligonucleotides, and wherein one or more cDNA molecules from the first cDNA sample anneal to the one or more oligonucleotides;

[0080] (iv) synthesizing one or more probe DNA molecules using the one or more oligonucleotides as primers and using the one or more cDNA molecules as templates;

[0081] (v) dissociating the one or more cDNA molecules from the one or more probe DNA molecules;

[0082] (vi) removing the first cDNA sample;

[0083] (vii) providing a second cDNA sample comprising double-stranded cDNA molecules;

[0084] (viii) denaturing the second cDNA sample to generate single-stranded cDNA molecules;

[0085] (ix) contacting a second cDNA sample with a surface, wherein one or more cDNA molecules from the second cDNA sample anneal to one or more probe DNA molecules;

[0086] (x) Extracting unannealed cDNA molecules for use as standard cDNA samples.

[0087] Standardization of nucleic acid samples leads to the generation of standardized nucleic acid samples. "Standardization" means that the level of RNA or cDNA sequences in the sample is more equal. To achieve this, the relative representativeness or level of less abundant sequences can be improved, and / or the relative representativeness or level of more abundant sequences can be reduced. In some specific embodiments, standardized RNA or cDNA comprises RNA or cDNA sequences with substantially the same level. For example, wherein the sequence level of standardized RNA or DNA varies less than 50%, less than 40%, less than 30%, less than 20% or less than 10%. Standardized RNA or cDNA can be a standardized RNA or cDNA sample in which at least a portion of the most abundant sequences in 10, 100, 1000 or 10000 kinds of the second RNA or cDNA sample has been removed. The method for processing nucleic acid described herein can be a method for balancing nucleic acid samples.

[0088] The method of the present invention can be used for both RNA and DNA. However, the use of double-stranded cDNA requires a denaturation step to produce single-stranded DNA molecules. Strand selection can also be used as part of double-stranded cDNA processing. Oligo dT molecules will only bind to cDNA strands containing poly (A) sequences.

[0089] Therefore, the method for processing cDNA may also comprise, after the last step (viii)):

[0090] (a) contacting the unannealed cDNA molecules with a second oligonucleotide array, wherein the second oligonucleotide array comprises two or more oligo-dT molecules attached to a surface, and wherein one or more of the cDNA molecules anneal to the oligo-dT molecules;

[0091] (b) removing unannealed cDNA molecules from the surface; and

[0092] (c) dissociating the annealed cDNA molecules from the oligo dT molecules to use as a treated cDNA sample.

[0093] According to all aspects of the present invention, in some specific embodiments, the oligonucleotide is a DNA molecule. According to all aspects of the present invention, in some specific embodiments, the oligonucleotide comprises an oligo dT sequence (optionally 2 to 200, 5 to 200, 2 to 100, 5 to 50, 7 to 25 or 12 to 18 nucleotides long). Therefore, in certain embodiments, the oligonucleotide is an oligo dT molecule. An oligo dT molecule means a molecule comprising a segment of deoxythymidine. An oligo dT molecule may have any length suitable for binding to a poly (A) tail (adenine nucleotide sequence) of a messenger RNA or a second strand of a double-stranded cDNA molecule. In certain embodiments, an oligo dT molecule is 2 to 100, 5 to 50, 7 to 25 or 12 to 18 nucleotides long. In other embodiments, an oligo dT molecule is at least 2, at least 5, at least 7, at least 12, at least 18 or at least 25 nucleotides long.

[0094] Oligonucleotides can be fixed on the surface. The surface can be two-dimensional, such as a glass slide, or three-dimensional, such as a microbead or a microsphere. According to all aspects of the present invention, in some specific embodiments, the surface is one or more beads or spheres, optionally magnetic beads. The method of the present invention can also be carried out in a microfluidic flow cell.

[0095] According to all aspects of the invention, in some embodiments, the RNA (the first RNA sample and / or the second RNA sample) comprises full-length RNA.

[0096] According to all aspects of the present invention, in some embodiments, the surface comprises two or more oligonucleotides, and the oligonucleotides are spaced apart ideally so that the DNA molecules it triggers do not interact with each other. Therefore, in certain embodiments, the oligonucleotides are spaced apart ideally so that the DNA (cDNA) molecules of the DNA array do not interact with each other. The ideal interval of a given sample type can be determined based on the length of the DNA (cDNA) molecules produced by the expectation. This is then determined by the (maximum) length of the RNA molecules in the first RNA sample or biological sample or the cDNA molecules in the first cDNA sample or the nucleic acid molecules in the (first) nucleic acid sample. In some specific embodiments, the interval between the oligonucleotides is at least 1 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times or at least 5 times of the (maximum) length of the RNA molecules in the first RNA sample or biological sample or the cDNA molecules in the first cDNA sample or the nucleic acid molecules in the (first) nucleic acid sample. The interval between oligonucleotides can be 1 to 5 times, 1.3 to 3.5 times, 1.4 to 3 times or 1.5 to 2.5 times of the (maximum) length of the RNA molecule in the first RNA sample or biological sample or the cDNA molecule in the first cDNA sample or the nucleic acid molecule in the (first) nucleic acid sample. In certain embodiments, the interval between oligonucleotides is 2 times of the (maximum) length of the RNA molecule in the first RNA sample or biological sample or the cDNA molecule in the first cDNA sample or the nucleic acid molecule in the (first) nucleic acid sample. In some specific embodiments, the interval between oligonucleotides is at least 2 times of the (maximum) length of the RNA molecule in the first RNA sample.

[0097] In other embodiments, the oligonucleotides are ideally spaced apart if the density of the oligonucleotides (of the oligonucleotide array) is between 0.01 oligonucleotides / 1 square micron and 10,000 oligonucleotides / 1 square micron, preferably between 0.1 oligonucleotides / 1 square micron and 1,000 oligonucleotides / 1 square micron, and more preferably between 1 oligonucleotide / 1 square micron and 100 oligonucleotides / 1 square micron.

[0098] In some embodiments, the first RNA sample and the second RNA sample are derived from the same (biological) sample. Similarly, the first cDNA sample and the second cDNA sample can be derived from the same (biological) sample. The first nucleic acid sample and the second nucleic acid sample can be derived from the same (biological) sample. Therefore, a part can be removed from a given sample, such as a blood sample (optionally treated to extract RNA), to form the first RNA sample, and another part can be removed to form the second RNA sample. Similarly, a part can be removed from a given sample, such as a blood sample (optionally treated to produce cDNA), to form the first cDNA sample, and another part can be removed to form the second cDNA sample. In addition, a part can be removed from a given sample, such as a blood sample (optionally treated to extract nucleic acid), to form the first nucleic acid sample, and another part can be removed to form the second nucleic acid sample. In other embodiments, the first RNA sample (or the first cDNA sample or the first nucleic acid sample) and the second RNA sample (or the second cDNA sample or the second nucleic acid sample) are derived from the same species, organism, tissue and / or cell type. In some embodiments, the first RNA sample (or first cDNA sample or first nucleic acid sample) and the second RNA sample (or second cDNA sample or second nucleic acid sample) are derived from blood.

[0099] According to all aspects of the invention, in some specific embodiments, the method further comprises sequencing the treated RNA, cDNA or nucleic acid. The method for treating nucleic acid (cDNA, RNA) can be a method for preparing nucleic acid (cDNA, RNA) for sequencing. Sequencing can be RNA or DNA sequencing. In certain embodiments, RNA is reverse transcribed into cDNA before sequencing. Sequencing can detect and / or quantify (target) nucleic acid molecules. Such a method comprises treating according to the invention, followed by sequencing of the treated product, optionally using a next generation sequencing (NGS) platform. Some examples of NGS platforms include Illumina sequencing (e.g., Hi-Seq and Mi-Seq), SMRT sequencing (Pacific Biosciences), nanopore sequencing, SoLID sequencing, pyrophosphate sequencing (e.g., Roche 454) and Ion-Torrent (Thermo Fisher), which are well known to technicians.

[0100] The present invention also relates to RNA extraction. Therefore, a method comprising:

[0101] (a) contacting a biological sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, wherein one or more RNA molecules from the biological sample anneal to the oligonucleotides in the oligonucleotide array;

[0102] (b) removing the unannealed sample from the surface; and

[0103] (c) dissociating the annealed RNA molecules from the oligonucleotides to obtain an RNA sample.

[0104] In a related aspect, there is provided a method comprising:

[0105] (a) contacting a biological sample with a surface, wherein the surface comprises one or more oligonucleotides, wherein one or more RNA molecules from the biological sample anneal to the one or more oligonucleotides;

[0106] (b) removing the unannealed sample; and

[0107] (c) dissociating the one or more RNA molecules from the one or more oligonucleotides to obtain an RNA sample.

[0108] These methods can be combined with other methods of the present invention to provide RNA samples. These methods can be performed before step (i) of the above method. In certain embodiments, a part of the RNA sample obtained forms a first RNA sample, and another part forms a second RNA sample. The RNA sample can be reverse transcribed into cDNA. In certain embodiments, the oligonucleotide comprises one or more oligo dT molecules. In some specific embodiments, the oligonucleotide is an oligo dT molecule. The oligo dT molecule will anneal with an mRNA molecule with a poly (A) tail. In other embodiments, the oligonucleotide comprises a random or unique sequence to capture a series of RNA in addition to mRNA. Custom oligonucleotides can be designed to capture specific target RNA molecules (with complementary sequences). If the RNA molecule does not comprise a poly (A) tail, it can be polyadenylated after extraction.

[0109] After extracting the treated RNA or cDNA, the method may further include dissociating the annealed RNA molecules from the cDNA molecules (or the annealed cDNA molecules from the DNA molecules). The dissociated molecules may be removed (optionally treated) to retain the surface containing the cDNA molecules (or DNA molecules). The surface may then be used to treat another RNA or cDNA sample. In some specific embodiments, the method for treating RNA further includes dissociating the annealed RNA molecules from the cDNA molecules and removing the dissociated RNA molecules from the surface after step (vi), and optionally, repeating steps (v) and (vi) with another RNA sample. In some specific embodiments, the method for treating cDNA further includes dissociating the annealed cDNA molecules from the DNA molecules and removing the dissociated cDNA molecules from the surface after step (viii), and optionally, repeating steps (vi), (vii) and (viii) with another cDNA sample.

[0110] According to all aspects of the present invention, in some specific embodiments, the length of oligonucleotide is at least 5 nucleotide, at least 10 nucleotide, at least 100 nucleotide, at least 200 nucleotide or at least 500 nucleotide.Oligonucleotide can be made up of 5 to 200 nucleotide.Oligonucleotide array or surface can comprise at least 10, at least 100, at least 1000, at least 10000, at least 100000 or at least 1 million oligonucleotides.The oligonucleotide that oligonucleotide array or surface comprise can be at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times, at least 1.9 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 100 times or at least 1000 times of the nucleic acid molecule in the RNA molecule in the first and / or the second RNA sample, the cDNA molecule in the first and / or the second cDNA sample or the nucleic acid sample. The oligonucleotide array or surface may comprise at least 10, at least 100, at least 1000, at least 10,000, at least 100,000 or at least 1 million oligonucleotides having unique sequences (i.e. no two sequences are identical). The oligonucleotides may comprise sequences complementary to the 10, 20, 50, 100, 1000 or 10,000 most abundant RNAs (mRNAs) in a given sample, optionally to the 10, 20, 50, 100, 1000 or 10,000 most abundant RNAs (mRNAs) in human blood. The oligonucleotides may comprise one or more sequences complementary to mRNA encoding human serum albumin, one or more alpha globulins (e.g. haptoglobin), one or more beta globulins (e.g. plasminogen) and / or one or more gamma globulins.

[0111] According to all aspects of the invention, in certain embodiments, the amount of RNA molecules in the (first and / or second) RNA sample or the amount of cDNA molecules in the (first and / or second) cDNA sample, or the amount of nucleic acid molecules in the (first and / or second) nucleic acid sample does not exceed the number of oligonucleotides in the oligonucleotide array and / or the number of DNA molecules in the DNA array. In some specific embodiments, the amount of RNA molecules in the second RNA sample does not exceed the number of cDNA molecules in the DNA array.

[0112] Biological sample and sample are used interchangeably in this article. According to all aspects of the present invention, in some specific embodiments, (biological) sample comprises biological fluid or fluid or lysate produced by biological material. Biological fluid may comprise blood. In some specific embodiments, blood is processed on the day of collection, no more than 72 hours after collection, no more than 2 weeks after collection, no more than 4 weeks after collection, or 4 to 12 months after collection. In certain embodiments, blood is stored at -80 ℃ before processing. Blood plasma and serum samples are envisioned. In some specific embodiments, sample is a human sample. Sample types include other biological fluids, such as saliva, urine or lymph. Other sample types include solid tissue, including frozen tissue or formalin-fixed paraffin embedded (FFPE) material. These samples can be processed to lyse cells.

[0113] RNA can be messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), long noncoding RNA (lncRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), small rDNA-derived RNA (srRNA), micro RNA (miRNA) or viral RNA, etc.

[0114] The present invention also relates to a system or a device for performing the method as described herein.

[0115] Thus, the present invention relates to an RNA processing device for producing processed RNA from a biological sample, the device comprising:

[0116] (i) a first module for receiving a biological sample, wherein the first module comprises:

[0117] (a) two or more oligonucleotides capable of annealing to one or more RNA molecules in a biological sample;

[0118] (b) a first waste outlet for removing unannealed sample;

[0119] (c) a sample outlet through which a portion of the sample comprising the one or more RNA molecules can flow after dissociation from the oligonucleotides; and

[0120] (ii) a second module for receiving one or more RNA molecules from the first module, wherein the second module comprises:

[0121] (a) two or more oligonucleotides capable of annealing to one or more RNA molecules in a sample;

[0122] (b) a processed RNA outlet, through which the processed RNA can be obtained;

[0123] (c) a waste RNA outlet for removing one or more RNA molecules;

[0124] The first module and the second module together define a flow path along which the sample can flow.

[0125] The oligonucleotide may comprise an oligo dT sequence (optionally 2 to 200, 5 to 200, 2 to 100, 5 to 50, 7 to 25 or 12 to 18 nucleotides long). Thus, in certain embodiments, the oligonucleotide of the first module and / or the oligonucleotide of the second module is an oligo dT molecule. An oligo dT molecule means a molecule comprising a stretch of deoxythymidine. An oligo dT molecule may have any length suitable for binding to the poly (A) tail (adenine nucleotide sequence) of a messenger RNA or the second strand of a double-stranded cDNA molecule. In certain embodiments, the oligo dT molecule is 2 to 100, 5 to 50, 7 to 25 or 12 to 18 nucleotides long.

[0126] In other embodiments, the oligonucleotides contain random or unique sequences to capture a range of RNAs in addition to mRNA.Custom oligonucleotides can be designed to capture specific target RNA molecules (with complementary sequences).

[0127] In certain embodiments, the oligonucleotides (oligo-dT molecules) of the first module are connected to the first surface, and the oligonucleotides (oligo-dT molecules) of the second module are connected to the second surface. The first module may also include a sample inlet, through which a biological sample can enter the first module. In some specific embodiments, the first module also includes a first reagent inlet, through which a reagent can enter the first module, and / or the second module also includes a second reagent inlet, through which a reagent can enter the second module. The RNA processing device may also include a temperature control device for regulating the temperature of the first module and / or the second module. In other embodiments, the first module includes a circulation cell and / or the second module includes a circulation cell. In some specific embodiments, the oligonucleotides are ideally spaced apart. Ideal spacing is discussed above. In some specific embodiments, the interval between the oligonucleotides of the first module and / or the second module is at least 2 times the (maximum) length of RNA molecules in the biological sample. In other embodiments, the oligonucleotides are ideally spaced apart if the density of the oligonucleotides (attached to the first and / or second surface) is between 0.01 oligonucleotides / 1 square micron and 10,000 oligonucleotides / 1 square micron, preferably between 0.1 oligonucleotides / 1 square micron and 1,000 oligonucleotides / 1 square micron, and more preferably between 1 oligonucleotide / 1 square micron and 100 oligonucleotides / 1 square micron.

[0128] According to all aspects of the present invention, in some specific embodiments, the RNA processing device further comprises a third module for receiving the processed RNA, wherein the third module comprises reagents for preparing the processed nucleic acid for sequencing. The RNA processing device may also comprise a fourth module for receiving the prepared RNA for sequencing, wherein the fourth module comprises sequencing reagents.

[0129] Another aspect of the invention provides use of an RNA processing device as described herein in a method for standardizing RNA.

[0130] The method for processing nucleic acid can be a method for removing nucleic acid from a sample. Thus, the method for processing RNA can be a method for removing (enriched) RNA from a second RNA sample. Similarly, the method for processing cDNA can be a method for removing (enriched) cDNA from a second cDNA sample.

[0131] In the case where a surface or oligonucleotide array comprises one or more oligonucleotides with a sequence complementary to the target nucleic acid of interest, the target nucleic acid can be combined with the one or more oligonucleotides. In this way, the target nucleic acid can be removed from the sample. The target nucleic acid can also be further processed, such as sequence checking.

[0132] Accordingly, the present invention provides a method for processing nucleic acids, the method comprising contacting a nucleic acid sample with a surface, wherein the surface comprises one or more oligonucleotides complementary to a target nucleic acid, wherein the one or more oligonucleotides are at least 100 nucleotides in length, and wherein the target nucleic acid anneals to the one or more oligonucleotides.

[0133] In some specific embodiments, the length of the oligonucleotide is at least 200 nucleotides, and optionally the length is at least 500 nucleotides. In certain embodiments, the surface comprises two or more oligonucleotides. In other embodiments, the oligonucleotide is connected to the surface.

[0134] According to all aspects of the invention, in some embodiments, the oligonucleotide complementary to the target nucleic acid is complementary to the entire length (or at least 70%, at least 80%, at least 90%) of the target nucleic acid.

[0135] The present invention also provides an RNA processing device for producing processed RNA from a biological sample, the device comprising:

[0136] (i) a first module for receiving a biological sample, wherein the first module comprises:

[0137] (a) two or more oligo-dT molecules capable of annealing to one or more RNA molecules in a biological sample;

[0138] (b) a first waste outlet for removing unannealed sample;

[0139] (c) a sample outlet through which a portion of the sample comprising the one or more RNA molecules can flow after dissociation from the two or more oligo-dT molecules; and

[0140] (ii) a second module for receiving one or more RNA molecules from the first module, wherein the second module comprises:

[0141] (a) one or more oligonucleotide molecules complementary to a target RNA in a sample;

[0142] (b) an unannealed sample outlet, which is used to remove the unannealed sample;

[0143] (c) a target RNA outlet through which the target RNA can be obtained after dissociation from the one or more oligonucleotide molecules;

[0144] The first module and the second module together define a flow path along which the sample can flow.

[0145] In certain embodiments, the one or more oligonucleotides complementary to a target RNA in a sample are at least 100 nucleotides in length, preferably at least 200 nucleotides in length, and more preferably at least 500 nucleotides in length.

[0146] The target nucleic acid may be from an RNA virus. The target nucleic acid may be (transcribed from) a bacterial gene, such as an antibiotic resistance gene. The target nucleic acid may be a biomarker for a disease.

[0147] The magnetic beads used in the claimed method can also be provided in the form of a kit. Therefore, in a related aspect, the present invention provides a kit for processing RNA samples, the kit comprising:

[0148] (a) one or more magnetic beads, wherein the one or more magnetic beads are linked to two or more oligo-dT molecules;

[0149] (b) hybridization buffer; and

[0150] (c) Reverse transcriptase.

[0151] Any suitable reverse transcriptase may be included in the kit. Suitable buffers are also well known and commercially available.

[0152] Another aspect of the invention provides the use of a kit as described herein in a method for normalizing RNA.

[0153] The present invention also provides a kit for processing a DNA sample, the kit comprising:

[0154] (a) one or more magnetic beads, wherein the one or more magnetic beads are linked to two or more oligo-dT molecules;

[0155] (b) hybridization buffer; and

[0156] (c) DNA polymerase.

[0157] Some examples of DNA polymerases include thermostable polymerases such as Taq or Pfu polymerase and various derivatives of these enzymes. Suitable buffers are also well known and commercially available.

[0158] Another aspect of the invention provides the use of a kit as described herein in a method for normalizing cDNA.

[0159] In another aspect, the present invention provides a kit for detecting a target nucleic acid in a sample, the kit comprising:

[0160] (a) one or more magnetic beads comprising one or more oligonucleotides complementary to a target nucleic acid, wherein the one or more oligonucleotides are at least 100 nucleotides in length; and

[0161] (b) Hybridization buffer.

[0162] The hybridization buffer may contain HEPES 1M (pH = 7.5), NaCl 5M and H 2 O. The kit of the present invention may also include dinucleotide triphosphate (dNTP), MgCl 2 and buffers, one or more, up to all.

[0163] The oligonucleotide may comprise a sequence complementary to the 10, 20, 50, 100, 1000 or 10000 most abundant RNAs (mRNAs) in a given sample, optionally a sequence complementary to the 10, 20, 50, 100, 1000 or 10000 most abundant RNAs (mRNAs) in human blood. The oligonucleotide may comprise one or more sequences complementary to mRNA encoding human serum albumin, one or more alpha globulins (e.g., haptoglobin), one or more beta globulins (e.g., plasminogen), and / or one or more gamma globulins.

[0164] Methods of RNA extraction and processing can be combined and incorporated into a pipeline for analyzing biological samples.

[0165] Accordingly, the present invention provides a method of analyzing a biological sample from a subject, the method comprising:

[0166] (a) extracting RNA from biological samples;

[0167] (b) preparing a processed RNA sample; and

[0168] (c) Sequencing the processed RNA.

[0169] The RNA can be a full-length RNA. The biological sample can include a biological fluid or a fluid or lysate produced by a biological material. In certain embodiments, the biological sample is a liquid biopsy. In some specific embodiments, the biological sample is a blood sample, optionally a human blood sample.

[0170] In certain embodiments, the preparation of processed RNA samples includes RNA standardization (reducing the variability of different RNA sequence levels in the sample). Therefore, the processed RNA sample can be a standardized RNA sample. "Standardization" means that the level of RNA sequence in the sample is more equal. In order to achieve this, the relative representativeness or level of less abundant sequences can be improved, and / or the relevant representativeness or level of more abundant sequences can be reduced. In some specific embodiments, the standardized RNA sample comprises RNA sequences with substantially the same level. For example, wherein the sequence level of the standardized RNA sample changes less than 50%, less than 40%, less than 30%, less than 20% or less than 10%. Standardized RNA can be a standardized RNA sample in which at least a portion of the most abundant sequences of 10, 100, 1000 or 10000 kinds in the sample has been removed.

[0171] Preparing the treated RNA sample may include balancing the RNA sample. Thus, in the treated RNA sample, the relative abundance of all unique RNA sequences may be more equal. For example, the levels of unique sequences in the treated RNA sample may vary by less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%.

[0172] In some specific embodiments, preparing a treated RNA sample reduces the variability of RNA levels (e.g., a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%). Preparing a treated RNA sample can achieve a more uniform distribution of RNA sequences. In a treated RNA sample, the difference in abundance between the most abundant RNA and the least abundant RNA can be reduced (e.g., a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%). In certain embodiments, preparing a treated RNA sample reduces the number of molecules (copy number) of the most abundant RNA molecules (1, 10, 100, 1000 or 10000) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. In some specific embodiments, the number of molecules (copy number) of the most abundant RNA molecule in the RNA sample is reduced by at least 50% in the treated RNA. In other embodiments, the relative abundance of the least abundant RNA molecule (1, 10, 100, 1000 or 10000) is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% in the treated RNA.

[0173] In certain embodiments, the processed RNA samples can be more easily analyzed. They can be sequenced more efficiently because the relative representation of less abundant sequences is increased.

[0174] In certain embodiments, the methods comprise diagnosing a disease in a subject.

[0175] Accordingly, the present invention provides a method for diagnosing a disease in a subject, the method comprising:

[0176] (a) extracting RNA from a biological sample from a subject;

[0177] (b) preparing a processed RNA sample; and

[0178] (c) Sequencing the processed RNA.

[0179] To make a diagnosis means to determine that a subject has a disease at the time of testing.

[0180] In other embodiments, the method comprises predicting a disease or identifying an increased risk of developing a disease. Accordingly, the present invention provides a method for predicting a disease or identifying an increased risk of developing a disease in a subject, the method comprising:

[0181] (a) extracting RNA from a biological sample from a subject;

[0182] (b) preparing a processed RNA sample; and

[0183] (c) Sequencing the processed RNA.

[0184] Prediction means determining that a subject who does not have a disease at the time of testing is at an increased risk of developing the disease. The increased risk can be a risk that is higher than the average risk of a population. The increased risk can be a risk that is higher than a pre-calculated threshold level. The threshold level can be a point above which the benefits of increased monitoring and / or preventive treatment outweigh the negative effects of potential unnecessary interventions. The increased risk can be a lifetime risk percentage greater than 1.5%, greater than 2%, greater than 5%, greater than 10%, greater than 50%, or greater than 75%.

[0185] In other embodiments, the methods include selecting a treatment for a subject having a disease, predicting the responsiveness of a subject having a disease to a therapeutic agent, and / or determining a clinical prognosis for a subject having a disease.

[0186] Processed RNA is sequenced to enable determination of the presence or absence and / or level of one or more RNA molecules. In certain embodiments, the presence or absence of one or more RNA molecules in the processed RNA sample is used to identify whether the subject suffers from a disease. In other embodiments, the level of one or more RNA molecules in the processed RNA sample is used to identify whether the subject suffers from a disease. Comparison with a reference point or reference value can be used to diagnose or predict clinical conditions or outcomes. Although as little as a specific RNA molecule (RNA molecule with a specific sequence) can be used to diagnose or predict clinical prognosis or the response to a therapeutic agent, using more RNA molecules (RNA molecules of other specific sequences) can improve specificity and sensitivity or diagnosis or prediction accuracy.

[0187] In certain embodiments, the RNA extracted from the sample comprises cell-free RNA.

[0188] In certain embodiments, extracting RNA from a biological sample comprises:

[0189] (a) contacting a biological sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, and wherein one or more RNA molecules from the sample anneal to the oligonucleotides in the oligonucleotide array;

[0190] (b) removing the unannealed sample from the surface; and

[0191] (c) dissociating the annealed RNA molecules from the oligonucleotides, thereby producing an RNA sample.

[0192] The oligonucleotide may comprise one or more oligo dT sequences. In some embodiments, the oligonucleotide is an oligo dT molecule.

[0193] Extracting RNA from a biological sample produces extracted RNA. In certain embodiments, preparing a processed RNA sample comprises following the steps of the method for processing RNA defined above. In other embodiments, preparing a processed RNA sample comprises taking a portion of the extracted RNA formed by extracting RNA from a biological sample as a first RNA sample, and taking a portion of the extracted RNA as a second RNA sample, and following the steps of the method for processing RNA defined above. Therefore, in some specific embodiments, preparing a processed RNA sample comprises taking a portion of the extracted RNA as a first RNA sample, and taking a portion of the extracted RNA as a second RNA sample, and:

[0194] (i) contacting a first RNA sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, and wherein two or more RNA molecules from the first RNA sample anneal to the oligonucleotides in the oligonucleotide array;

[0195] (ii) extending two or more of the oligonucleotides by reverse transcription using the annealed RNA molecules as templates to generate a DNA array comprising two or more cDNA molecules;

[0196] (iii) dissociating the annealed RNA molecules from the cDNA molecules;

[0197] (iv) removing the first RNA sample from the surface;

[0198] (v) contacting a second RNA sample with the DNA array, wherein one or more RNA molecules from the second RNA sample anneal to the cDNA molecules; and

[0199] (vi) extracting the unannealed RNA molecules, thereby producing processed RNA.

[0200] In other embodiments, preparing the processed RNA sample comprises:

[0201] (i) contacting the extracted RNA with an oligonucleotide array (e.g., a DNA, optionally a cDNA array), wherein one or more RNA molecules from the extracted RNA anneal to one or more oligonucleotides in the oligonucleotide array; and

[0202] (ii) extracting the unannealed RNA molecules, thereby producing processed RNA.

[0203] A method of analyzing a biological sample from a subject may include using one or more of the RNA processing devices and kits of the invention.

[0204] In some embodiments, sequencing the processed RNA comprises long-read sequencing.

[0205] Another aspect of the invention provides the use of a method, apparatus or kit as described herein in an RNA or DNA sequencing process (optionally for discovering new RNAs and / or detecting low-abundance RNAs), and optionally wherein the sequencing is single-cell sequencing.

[0206] Another aspect of the present invention provides use of the method, device or kit as described herein in a metagenomic sequencing process for discovering new microorganisms and / or detecting low-abundance microorganisms.

[0207] Another aspect of the invention provides the use of a method, device or kit as described herein in screening a DNA or RNA sample or screening a genetic sample for the presence of an infectious disease.

[0208] Another aspect of the invention provides the use of a method, device or kit as described herein in the detection of a nucleic acid biomarker, optionally a disease biomarker, and optionally a cancer biomarker.

[0209] In some embodiments, according to all aspects of the invention, the method further comprises reporting a result. The result can be in the form of an RNA or DNA sequence, an indication of the presence or absence of a microorganism or disease, and / or an indication of the presence or absence or level of a disease biomarker. BRIEF DESCRIPTION OF THE DRAWINGS

[0210] The above and other aspects of the present invention will now be described in further detail, by way of example only, with reference to the following examples and accompanying drawings.

[0211] Figure 1 - Schematic representation of magnetic beads with attached oligo dT primers. During RNA processing according to the invention, RNA molecules are annealed to oligo dT molecules and reverse transcribed to produce cDNA copies attached to the beads.

[0212] Figure 2 - Schematic diagram of magnetic beads with cDNA probes attached. The magnetic beads are reintroduced to another batch of RNA. A portion of the RNA is annealed to the probe (captured RNA). The beads are then fixed and a solution containing the standardized RNA is extracted.

[0213] Figure 3 - Schematic diagram of a microfluidic flow cell for RNA processing according to the present invention. RNA flows through and the temperature is cooled down to allow poly-A to anneal to the oligo-dT forest.

[0214] Figure 4 - Schematic diagram of a microfluidic flow cell for RNA processing according to the present invention. Reverse transcription material is added and incubated for reverse transcription.

[0215] Figure 5 - Schematic diagram of a microfluidic flow cell for RNA treatment according to the invention. Heating is performed to dissociate the RNA, which is then flushed out.

[0216] Figure 6 - Schematic diagram of a microfluidic flow cell for RNA processing according to the invention showing a cDNA forest prepared for RNA to be normalized.

[0217] Figure 7 - Schematic diagram of a microfluidic flow cell for RNA treatment according to the invention. New RNA is added and incubated between 45°C and 75°C (e.g. at about 68°C) for association. Enriched RNA is annealed to the cDNA forest, while free normalized RNA flows through.

[0218] Figure 8 ——Schematic diagram of a microfluidic flow cell for RNA treatment according to the present invention. Heating to 80°C to 100°C (e.g. 98°C) causes RNA to dissociate and flow through to waste. Then the flow can be repeated from Figure 7 Start the cycle.

[0219] Fig. 9 ——Schematic diagram of RNA extraction according to the present invention. Sample with lysed cells flows over the surface. The temperature is cooled down to allow poly-A to anneal with oligo dT. The flow cell is rinsed to retain only bound RNA. Heating is performed until RNA is dissociated and flows through to the next step.

[0220] Fig.10 - Schematic diagram of an RNA processing device according to the present invention.

[0221] Fig.11 - Schematic diagram of RNA processing according to the invention, wherein the oligonucleotides attached to the surface are complementary to the target RNA (designed probe cDNA forest). The sample with lysed cells is flowed through. Incubation between 45°C and 75°C (e.g. at about 68°C) allows full-length association. The cells are rinsed, leaving only the bound RNA. Heating is performed until the RNA is dissociated, and flowed through for further processing.

[0222] Fig.12 ——Schematic diagram of DNA processing according to the present invention, wherein the oligonucleotides attached to the surface are complementary to the target DNA (designed probe cDNA forest). The sample with lysed cells and fragmented DNA flows through. Heating to 80°C to 100°C (e.g. 98°C) dissociates the double strands. Incubation between 45°C and 75°C (e.g. at about 68°C) allows full-length association. The cells are rinsed, leaving only the bound DNA. Heating is performed until the DNA is dissociated and flows through for further processing. DETAILED DESCRIPTION

[0223] Figure 1Schematically illustrates an array of oligonucleotides (in this case, oligo dT molecules) connected to magnetic beads. The first RNA sample is contacted with the magnetic beads, and the RNA molecules containing the poly-A tail are annealed with the oligo dT molecules. Using the annealed RNA molecules as templates, the oligo dT molecules are extended by reverse transcription to produce cDNA molecules (DNA arrays) connected to the beads. The abundant RNA molecules (the RNA sequences that occur more frequently in the sample) will produce more cDNA molecules. The annealed RNA molecules are dissociated from the cDNA molecules, and the first RNA sample is removed from the magnetic beads, retaining the cDNA molecules connected to the beads.

[0224] This stage of the method for generating bead-linked cDNA molecules (DNA array) involves the following steps:

[0225] (i) adding magnetic beads having oligo dT molecules to a purified RNA solution (first RNA sample);

[0226] (ii) heating to 65°C to 100°C (above 70°C, optionally 70°C to 100°C, 80°C to 100°C or 90°C to 100°C) for 5 seconds to 1 minute to remove secondary structures;

[0227] (iii) cooling to below 65° C. (i.e., 65° C. or lower, e.g., 30° C. to 65° C.) for 5 seconds to 1 minute to allow the oligo dT molecule to anneal to the poly-A tail of the RNA;

[0228] (iv) adding reverse transcription materials (reverse transcriptase, dNTP, etc.);

[0229] (v) incubating for reverse transcription (at 30° C. to 80° C., optionally at 30° C. to 60° C., for 1 minute to 2 hours);

[0230] (vi) heating to 80° C. to 100° C. (e.g., about 98° C.) for 5 seconds to 1 minute to dissociate the RNA from the cDNA copies;

[0231] (vii) immobilizing the beads using an external magnet;

[0232] (viii) Removing the liquid solution containing RNA.

[0233] The second RNA sample is then contacted with the beads containing the attached cDNA molecules. Figure 2 As shown in , RNA molecules from the second RNA sample are annealed to cDNA molecules having complementary sequences. Figure 1The stages shown in FIG. 1 produce more cDNA molecules, so more abundant RNA molecules in the second RNA sample will be captured by the cDNA molecules, then the less abundant RNA molecules will be captured. As a result, the RNA molecules that do not anneal to the cDNA molecules have a more uniform sequence distribution—the RNA is normalized because it is no longer dominated by a few very abundant sequences. The magnetic beads are then immobilized and the non-annealed RNA molecules are extracted, thereby producing the processed RNA.

[0234] For each reaction cycle, the amount of RNA molecules in the second RNA sample should ideally not exceed the number of DNA molecules in the DNA array (cDNA forest). If the number of DNA arrays exceeds each pass of RNA, it ensures that there are enough probes to anneal to high-abundance RNA.

[0235] This stage of the method for producing processed RNA comprises the following steps:

[0236] (i) adding a new aliquot of the RNA solution (second RNA sample), optionally from the same sample;

[0237] (ii) heating to 65°C to 100°C (above 70°C, optionally 70°C to 100°C, 80°C to 100°C or 90°C to 100°C) for 5 seconds to 1 minute to remove secondary structures;

[0238] (iii) cooling and incubating at between 45°C and 75°C (e.g., at about 68°C) for 10 seconds to 8 hours to allow controlled full-length association with the cDNA;

[0239] (iv) immobilizing the magnetic beads using an external magnet;

[0240] (v) extracting a liquid solution containing the processed (normalized) RNA fraction;

[0241] (vi) Repeat with additional aliquots of the RNA solution to obtain more treated RNA (after dissociation of the annealed RNA).

[0242] The invention enables normalization of full-length RNA. Advantages of directly analyzing RNA include the fact that no PCR is required (saving time and reagents, and no PCR artefacts), there is no bias, and nanopore sequencing can directly detect modifications present in RNA (modifications change the way the RNA moves through the pore).

[0243] The oligonucleotide array may be attached to any suitable surface, and the invention is not limited to the use of magnetic beads.For example, the method may also be performed in a microfluidic flow cell.

[0244] Figure 3Schematically showing an array of oligonucleotides, in this case oligo dT molecules (also referred to herein as oligo dT forests), attached to the surface of a flow cell. A first RNA sample flows through the flow cell and anneals RNA molecules containing poly-A tails to the oligo dT molecules. The temperature is cooled to below 65°C (i.e., 65°C or below, optionally 30°C to 65°C) to anneal the oligo dT molecules to the poly-A tails of the RNA.

[0245] Add reverse transcription reagents and incubate. Figure 4 As shown in , using annealed RNA molecules as templates, oligo dT molecules are extended by reverse transcription to produce cDNA molecules (DNA arrays) attached to the surface via oligo dT sequences. Abundant RNA molecules (RNA sequences that appear more frequently in the sample) will produce more cDNA molecules.

[0246] like Figure 5 As shown in, the annealed RNA molecules are dissociated from the cDNA molecules by heating. The first RNA sample is then rinsed out, and the cDNA molecules (DNA array or cDNA forest) connected to the surface are retained, as shown in FIG. Figure 6 as shown in .

[0247] The second RNA sample is then contacted with the surface containing the cDNA molecules and incubated at about 68°C. Figure 7 As shown in , RNA molecules from the second RNA sample are annealed with cDNA molecules having complementary sequences. Figure 4 In the steps shown in , the abundant RNA molecules produce more cDNA molecules, so more abundant RNA molecules in the second RNA sample will be captured by the cDNA molecules, then the less abundant RNA molecules will be captured. As a result, the RNA molecules that do not anneal to the cDNA molecules have a more uniform sequence distribution - the RNA is normalized because it is no longer dominated by a few very abundant sequences. The unannealed RNA molecules flow through, resulting in processed RNA.

[0248] Figure 8 Another step is shown to dissociate the annealed RNA molecules from the cDNA molecules by heating to 98° C. The dissociated RNA flows through to waste. The surface containing the cDNA molecules can then be reused with additional RNA samples to produce more processed RNA.

[0249] like Fig. 9As shown in, similar principles can be applied to RNA extraction. A biological sample with lysed cells containing RNA, DNA, protein, etc. flows through an array of oligonucleotides connected to the surface of a circulation pool, in this case oligo dT molecules (also referred to herein as oligo dT forests). The temperature is cooled to below 65°C (i.e., 65°C or below 65°C, optionally 30°C to 65°C) to anneal the oligo dT molecules to the poly-A tail of the RNA. The circulation pool is rinsed, retaining only the annealed RNA. The temperature is then raised to 80°C to 100°C (e.g., 98°C) to dissociate the annealed RNA molecules from the oligonucleotides to obtain an RNA sample. The RNA then flows through for further processing.

[0250] RNA extraction and RNA processing can be connected by combining microfluidic flow cells. One flow cell (also referred to herein as a module or reaction chamber) extracts RNA, which is then processed in another flow cell (or module). Fig.10 Schematically shows an RNA processing device, which includes two flow cells. The biological sample is input through the sample inlet in the first flow cell. The biological sample can be a sample of lysed cells containing RNA, DNA, protein, etc. The reagent is introduced through the reagent inlet, such as a buffer and / or an RNA stabilizing reagent. The surface of the first flow cell is as shown in FIG. Fig. 9 As shown in, i.e., an array of oligonucleotides (in this case, oligo dT molecules) connected to the surface of the circulation cell. Both the first circulation cell and the second circulation cell include a temperature control device (thermocontrol) for regulating the temperature. The temperature control device allows the temperature to be cooled to less than 65 ° C (i.e., 65 ° C or less than 65 ° C, optionally 30 ° C to 65 ° C) to anneal the oligonucleotides to the RNA. The first circulation cell includes a first waste outlet for removing unannealed samples so that only annealed RNA is retained when the first circulation cell is rinsed. Then, the temperature control device allows the temperature to be increased to 80 ° C to 100 ° C (e.g., 98 ° C) to dissociate the annealed RNA molecules from the oligonucleotides, thereby obtaining RNA samples. The first circulation cell includes a sample outlet, and the RNA sample can flow through the sample outlet after dissociating with the oligonucleotides. The first circulation cell and the second circulation cell together define a flow path along which the sample can flow. Therefore, the first and second circulation cells are connected by a connector (e.g., a tube), which allows RNA to flow from the first circulation cell to the second circulation cell for further processing.

[0251] The RNA sample enters through the RNA sample inlet in the second circulation cell. The second circulation cell (module) also includes a second reagent inlet, through which reagents can enter the second circulation cell. The surface of the second circulation cell includes an array of oligonucleotides (e.g., oligo dT molecules) connected to the circulation cell surface. The RNA sample flows through the circulation cell and anneals the RNA molecules to the oligonucleotides. Reverse transcription reagents are added through the second reagent inlet. Using the annealed RNA molecules as templates, the oligonucleotides are extended by reverse transcription to produce cDNA molecules (DNA arrays) connected to the surface. The annealed RNA molecules are dissociated from the cDNA molecules by heating using a temperature control device. Therefore, the temperature control device can heat the RNA molecules to 98°C. The second circulation cell includes a waste RNA outlet to remove one or more RNA molecules. The waste RNA outlet allows the RNA sample to be rinsed out, retaining the cDNA molecules connected to the surface.

[0252] Then, the additional RNA sample enters the second flow cell, contacts the surface containing the attached cDNA molecules, and is incubated between 45° C. and 75° C. (e.g., at about 68° C.). The RNA molecules from the additional RNA sample are annealed to the cDNA molecules having complementary sequences. The second flow cell comprises a treated RNA outlet through which the unannealed RNA molecules flow, thereby producing treated (normalized) RNA.

[0253] In the case where the surface or oligonucleotide array comprises one or more oligonucleotides with a sequence complementary to the target nucleic acid of interest, the target nucleic acid can be combined with the one or more oligonucleotides. In this way, the target nucleic acid can be removed from the sample. The target nucleic acid can also be further processed, such as sequencing. The RNA processing device discussed above may comprise an additional flow cell, which comprises one or more oligonucleotides with a sequence complementary to the target nucleic acid of interest. Alternatively, the second flow cell may comprise one or more oligonucleotides with a sequence complementary to the target nucleic acid of interest.

[0254] Target nucleic acids can also be extracted directly from biological samples. Fig.11A surface or array comprising oligonucleotides complementary to a target RNA (also referred to herein as a designed probe cDNA forest) is shown. The length of the oligonucleotide is at least 100 nucleotides. A biological sample having lysed cells comprising RNA, DNA, protein, etc. flows through an array of oligonucleotides. Incubation between 45°C and 75°C (e.g., at about 68°C) allows the full-length association of the target RNA with the oligonucleotide to be performed. The flow cell is rinsed to retain only the annealed RNA. The temperature is then raised to 80°C to 100°C (e.g., 98°C) to dissociate the annealed RNA molecules from the oligonucleotides, thereby obtaining the target RNA. The RNA is then flowed through for further processing. The remaining RNA, DNA, and protein can then be discarded or further processed.

[0255] The methods, devices and kits described herein (when used to process RNA) can be used for RNA biomarkers targeting RNA viruses, bacterial genes (e.g., antibiotic resistance genes) and diseases. This is combined with RNA sequencing to allow accurate diagnosis. DNA arrays (probe forests) are reusable. Therefore, if combined with (nanopore) sequencing or other detection methods (PCR, LAMP, etc.), the device can be used as a fast, reusable screening of viral infections. The methods, kits and devices can also be used in agricultural technology (agritech) to monitor diseases of crops and livestock. Sample processing is fast and efficient.

[0256] The methods, devices and kits described herein can also be used to process DNA. However, the use of double-stranded DNA requires a denaturation step to generate single-stranded DNA molecules. Fig.12 A surface or array comprising oligonucleotides complementary to a target DNA (also referred to herein as a designed probe cDNA forest) is shown. The length of the oligonucleotide is at least 100 nucleotides. A biological sample having lysed cells comprising RNA, fragmented DNA, proteins, etc. flows through an array of oligonucleotides. The sample is heated to 80°C to 100°C (e.g., 98°C) to dissociate the double-stranded DNA. Then, incubate between 30°C and 75°C (e.g., at about 68°C) to allow the full-length association of the target DNA with the oligonucleotide to be performed. The flow cell is rinsed to retain only the annealed DNA. The temperature is then raised to 80°C to 100°C (e.g., 98°C) to dissociate the annealed DNA molecules from the oligonucleotides to obtain the target DNA. Then, the target DNA flows through for further processing. The remaining RNA, DNA, and protein can then be discarded or further processed.

[0257] The methods, devices and kits described herein (when used to process DNA) can be used for targeting DNA viruses, for bacterial identification (identification of other microorganisms), for detecting DNA biomarkers of diseases, and for rapid DNA identification as a means of verifying individual identity. DNA arrays (probe forests) are reusable. Therefore, if combined with (nanopore) sequencing or other detection methods (PCR, LAMP, etc.), the device can be used as a rapid and reusable screening of viral infections. The methods, kits and devices can also be used in agricultural technology to monitor diseases of crops and livestock. Sample processing is fast and efficient.

[0258] Where an oligonucleotide complementary to a target nucleic acid is used, the oligonucleotide may be complementary to the entire length (or at least 70%, at least 80% or at least 90%) of the target nucleic acid. This differs from typical probe-based systems that use only short oligonucleotide sequences to target nucleic acids.

[0259] In DNA array (cDNA forest), there is an ideal distance between DNA molecules so that they do not interact with each other. This distance is affected by the expected cDNA length, so it is ideal that any two points need to be approximately twice the longest cDNA length. For example, when the biological sample is (people) blood, the maximum length of RNA is about 5kb, so the maximum length of cDNA produced thereby will be about 5kb. Therefore, at least 10kb (6000nm) will be the ideal interval between the cDNA molecules in the oligonucleotide array and / or the DNA array. In the case of using an oligonucleotide complementary to the target DNA or RNA, the distance between the oligonucleotide can be smaller, because known sequences allow the design of oligonucleotide sequences, and therefore there is minimal interaction. Therefore, in the case of using an oligonucleotide complementary to the target DNA or RNA, the distance between the oligonucleotide can be at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times, at least 1.8 times or at least 1.9 times of the oligonucleotide length.

[0260] As mentioned above, the density of oligonucleotides in the oligonucleotide array affects the density of DNA molecules in the DNA array. Therefore, a means to prevent the DNA molecules in the DNA array from interacting with each other is to use the oligonucleotides at a certain interval (i.e., maximum density) in the array, as discussed above. The density of DNA molecules in the DNA array is also affected by the concentration of RNA or cDNA molecules in the first RNA or the first cDNA sample respectively. When appropriate, how many oligonucleotides are there in this concentration impact oligonucleotide array to capture RNA molecules or cDNA molecules. This in turn affects how many DNA molecules are synthesized using captured RNA or DNA as template. Therefore, the concentration of RNA or cDNA molecules in the adjustable first RNA or the first cDNA sample is to prevent the DNA molecules in the DNA array from interacting with each other.

[0261] Performance is also based on the ratio between RNA and DNA arrays (cDNA forests). Thermal and kinetic control are associated with optimal performance. Micropumps can be used to generate laminar or turbulent flow.

[0262] The method of RNA extraction and processing as described above can be combined and incorporated into a process for analyzing biological samples. The method comprises:

[0263] (a) extracting RNA from biological samples;

[0264] (b) preparing a processed RNA sample; and

[0265] (c) Sequencing the processed RNA.

[0266] A blood or other liquid biopsy sample is collected from a subject in a container with a cell lysis buffer and an RNA stabilizing reagent. RNA stabilizing reagents are commercially available and include (Sigma-Aldrich) and RNAprotect (Qiagen). Suitable buffers contain EDTA, sodium citrate and ammonium sulfate. Incubation for cell lysis can be, for example, 1 minute to 3 hours. The sample is then added to the RNA processing device as described above.

[0267] RNA extraction (purification) is first performed. The first reaction chamber (also referred to herein as a circulation cell or module) uses a solution of oligonucleotides to purify RNA, which can be oligo dT or random sequences. These oligonucleotides bind to the chamber surface to form an oligo forest. After the sample solution is pumped into the first chamber, the chamber is heated to a temperature of 30°C to 75°C (optionally 30°C to 65°C or 60°C to 65°C) to allow RNA to anneal to the oligo forest. After the incubation period, the remaining fluid is flushed out to the waste channel. The chamber is then heated to above 75°C (e.g., 80°C to 100°C) to release the remaining annealed RNA. It is then pumped through to the second reaction chamber.

[0268] The next step is to prepare a processed RNA sample, in this case RNA standardization. The second reaction chamber (circulation cell, module) has an additional oligo forest. The purified RNA is cooled to below 65°C (i.e. 65°C or below 65°C, for example 30°C to 65°C), optionally below 60°C, in the chamber to allow annealing with the oligo forest. Reverse transcriptase and buffer are then added to generate complementary DNA chains using the oligo forest as primers. Once reverse transcription is complete, the chamber is heated to 80°C to 100°C (optionally above 90°C) to dissociate the RNA from the cDNA forest. The solution is then rinsed into the waste channel. The additional purified RNA sample is then pumped into the second chamber with the cDNA forest. The chamber is heated to 45°C to 75°C (optionally 60°C to 75°C) to allow full-length annealing of the RNA to the cDNA.

[0269] After incubation, the unannealed RNA is pumped into the collection chamber for further processing (to be sequenced). The second chamber is then heated to 80°C to 100°C (optionally above 90°C) to release the RNA. The dissociated RNA is flushed to the waste channel. This process is repeated until a sufficient amount of standardized RNA is generated for sequencing.

[0270] The next stage is preparation for sequencing. Then, the standardized RNA can be pumped into another reaction chamber (circulation cell, module), which will prepare the sequencing library. Depending on the sequencing technology, this may involve the connection of adapters, second strand synthesis and / or any other modification required to allow sequencing. Then, the sequencing library will be pumped into the sequencing chamber for sequencing.

[0271] The next stage is sequencing and data processing. During sequencing, the raw data can be uploaded to a cloud server for data processing and archiving.

[0272] Combining RNA extraction and processing in this manner provides a device that can be used for immediate processing of blood or other samples, thereby minimizing problems with RNA degradation.

[0273] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications to the present invention, in addition to those described herein, will also become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. In addition, all embodiments described herein are considered to be widely applicable and may be appropriately combined with any and all other consistent embodiments.

[0274] Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. A method for processing RNA, comprising: (i) contacting a first RNA sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, and wherein two or more RNA molecules from the first RNA sample anneal to the oligonucleotides in the oligonucleotide array; (ii) extending two or more of the oligonucleotides by reverse transcription using the annealed RNA molecules as templates to generate a DNA array comprising two or more cDNA molecules; (iii) dissociating the annealed RNA molecule from the cDNA molecule; (iv) removing the first RNA sample from the surface; (v) contacting a second RNA sample with the DNA array, wherein one or more RNA molecules from the second RNA sample anneal to the cDNA molecules; and (vi) extracting the unannealed RNA molecules, thereby producing processed RNA.

2. A method for processing cDNA, comprising: (i) denaturing a first cDNA sample comprising double-stranded cDNA molecules to generate single-stranded cDNA molecules; (ii) contacting the first cDNA sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, and wherein two or more cDNA molecules from the first cDNA sample anneal to the oligonucleotides in the oligonucleotide array; (iii) extending two or more of the oligonucleotides using the annealed cDNA molecules as templates to generate a DNA array comprising two or more DNA molecules; (iv) dissociating the annealed cDNA molecules from the DNA molecules in the DNA array; (v) removing the first cDNA sample from the surface; (vi) denaturing the second cDNA sample comprising double-stranded cDNA molecules to generate single-stranded cDNA molecules; (vii) contacting the second cDNA sample with the DNA array, wherein one or more cDNA molecules from the second cDNA sample anneal to the DNA molecules; (viii) extracting the unannealed cDNA molecules to generate processed cDNA.

3. The method of claim 2, further comprising the following step (viii): (a) contacting the unannealed cDNA molecules with a second oligonucleotide array, wherein the second oligonucleotide array comprises two or more oligo-dT molecules attached to a surface, and wherein one or more of the cDNA molecules anneal to the oligo-dT molecules; (b) removing unannealed cDNA molecules from the surface; and (c) dissociating the annealed cDNA molecules from the oligo dT molecules to use as a treated cDNA sample.

4. The method of any one of claims 1 to 3, wherein the oligonucleotide comprises an oligo dT sequence.

5. The method of any one of claims 1 to 4, wherein the surface is one or more magnetic beads.

6. The method of any one of claims 1 to 5, wherein the method is performed in a microfluidic flow cell.

7. The method of any one of claims 1 or 4 to 6, wherein the first RNA sample and / or the second RNA sample comprises full-length RNA.

8. The method of any one of claims 1 to 7, wherein the oligonucleotides are ideally spaced apart so that the DNA (cDNA) molecules of the DNA array do not interact with each other.

9. The method of any one of claims 1 to 8, wherein the first RNA sample and the second RNA sample (or the first cDNA sample and the second cDNA sample) are derived from the same sample.

10. The method of any one of claims 1 to 9, further comprising sequencing the treated RNA or processed cDNA.

11. The method according to any one of claims 1 to 10, further comprising before step (i): (a) contacting a biological sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, and wherein one or more RNA molecules from the biological sample anneal to the oligonucleotides in the oligonucleotide array; (b) removing the unannealed sample from the surface; and (c) dissociating the annealed RNA molecules from the oligonucleotides to obtain an RNA sample.

12. The method of any one of claims 1 or 4 to 11, further comprising dissociating the annealed RNA molecules from the cDNA molecules after step (vi) and removing the dissociated RNA molecules from the surface, and optionally, repeating steps (v) and (vi) with additional RNA samples.

13. The method of claim 11 or claim 12, wherein the biological sample comprises a biological fluid or a fluid or lysate produced from a biological material.

14. An RNA processing device for producing processed RNA from a biological sample, the device comprising: (i) a first module for receiving the biological sample, wherein the first module comprises: (a) two or more oligonucleotides capable of annealing to one or more RNA molecules in the biological sample; (b) a first waste outlet for removing unannealed sample; (c) a sample outlet through which a portion of the sample comprising the one or more RNA molecules can flow after dissociation from the oligonucleotide; and (ii) a second module for receiving the one or more RNA molecules from the first module, wherein the second module comprises: (a) two or more oligonucleotides capable of annealing to one or more RNA molecules in a sample; (b) a processed RNA outlet, through which the processed RNA can be obtained; (c) a waste RNA outlet for removing one or more RNA molecules; The first module and the second module together define a flow path along which a sample can flow. 15 . The RNA processing device of claim 14 , wherein the oligonucleotides of the first module are attached to a first surface, and the oligonucleotides of the second module are attached to a second surface. 16 . The RNA processing device according to claim 14 or claim 15 , wherein the first module further comprises a sample inlet, and the biological sample can enter the first module through the sample inlet.

17. The RNA processing device according to any one of claims 14 to 16, wherein the first module further comprises a first reagent inlet, through which a reagent can enter the first module, and / or wherein the second module further comprises a second reagent inlet, through which a reagent can enter the second module.

18. The RNA processing device according to any one of claims 14 to 17, further comprising a temperature control device for adjusting the temperature of the first module and / or the second module.

19. The RNA processing device according to any one of claims 14 to 18, wherein the first module comprises a flow cell and / or the second module comprises a flow cell.

20. The RNA processing device of any one of claims 14 to 19, wherein the oligonucleotides are ideally spaced.

21. A method for processing nucleic acids, the method comprising contacting a nucleic acid sample with a surface, wherein the surface comprises one or more oligonucleotides complementary to a target nucleic acid, wherein the one or more oligonucleotides are at least 100 nucleotides in length, and wherein the target nucleic acid anneals to the one or more oligonucleotides.

22. The method of claim 21, wherein the one or more oligonucleotides are at least 200 nucleotides in length, optionally at least 500 nucleotides in length.

23. An RNA processing device for producing processed RNA from a biological sample, the device comprising: (i) a first module for receiving the biological sample, wherein the first module comprises: (a) two or more oligo-dT molecules capable of annealing to one or more RNA molecules in the biological sample; (b) a first waste outlet for removing unannealed sample; (c) a sample outlet through which a portion of the sample comprising the one or more RNA molecules can flow after dissociation from the two or more oligo-dT molecules; and (ii) a second module for receiving the one or more RNA molecules from the first module, wherein the second module comprises: (a) one or more oligonucleotide molecules complementary to a target RNA in a sample; (b) an unannealed sample outlet, which is used to remove the unannealed sample; (c) a target RNA outlet, through which the target RNA is accessible after dissociation from the one or more oligonucleotide molecules; The first module and the second module together define a flow path along which a sample can flow.

24. The RNA processing device of claim 23, wherein the one or more oligonucleotides complementary to the target RNA in the sample are at least 100 nucleotides in length, preferably at least 200 nucleotides in length, and more preferably at least 500 nucleotides in length.

25. A kit for processing RNA samples, the kit comprising: (a) one or more magnetic beads, wherein the one or more magnetic beads are linked to two or more oligo-dT molecules; (b) hybridization buffer; and (c) Reverse transcriptase.

26. A kit for processing a DNA sample, the kit comprising: (a) one or more magnetic beads, wherein the one or more magnetic beads are linked to two or more oligo-dT molecules; (b) hybridization buffer; and (c) DNA polymerase.

27. A kit for detecting a target nucleic acid in a sample, the kit comprising: (a) one or more magnetic beads comprising one or more oligonucleotides complementary to the target nucleic acid, wherein the one or more oligonucleotides are at least 100 nucleotides in length; and (b) Hybridization buffer.

28. The kit of any one of claims 26 to 28, further comprising one or more, up to all, of dinucleotide triphosphates (dNTPs), MgCl2, and a buffer.

29. A method of analyzing a biological sample from a subject, the method comprising: (a) extracting RNA from the biological sample; (b) preparing a processed RNA sample; as well as (c) sequencing the processed RNA.

30. The method of claim 29, wherein the RNA is a full-length RNA.

31. The method of claim 29 or claim 30, wherein the biological sample comprises a biological fluid or a fluid or lysate produced from a biological material, optionally wherein the biological fluid comprises a blood sample.

32. The method of any one of claims 29 to 31, wherein the method comprises diagnosing a disease in the subject.

33. The method of any one of claims 29 to 32, wherein the RNA extracted from the sample comprises cell-free RNA.

34. The method of claim 32 or 33, wherein the presence or absence of one or more RNA molecules in the processed RNA sample is used to identify whether the subject has the disease.

35. The method of any one of claims 29 to 34, wherein extracting RNA from the biological sample comprises: (a) contacting the biological sample with an oligonucleotide array, wherein the oligonucleotide array comprises two or more oligonucleotides attached to a surface, and wherein one or more RNA molecules from the sample anneal to the oligonucleotides in the oligonucleotide array; (b) removing the unannealed sample from the surface; and (c) dissociating the annealed RNA molecules from the oligonucleotides, thereby producing an RNA sample.

36. The method of claim 35, wherein the oligonucleotide comprises one or more oligo-dT sequences.

37. The method of any one of claims 29 to 36, wherein preparing the treated RNA sample comprises taking a portion of the extracted RNA as a first RNA sample, and taking a portion of the extracted RNA as a second RNA sample, and following the steps of the method of any one of claims 1 or 4 to 10.

38. The method of any one of claims 29 to 37, comprising using the RNA processing device of any one of claims 14 to 20, 23 or 24 or the kit of any one of claims 25 to 28.

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