Methods for selectively quantifying RNA in a biological sample

Abstract

The present invention is in the field of nucleic acid detection and quantification and relates to a method for detecting and quantifying target RNA sequences in a sample, the method comprising the steps of: a) reverse transcribing RNA present in a biological sample into complementary DNA (cDNA) using a plurality of oligonucleotides, wherein the oligonucleotides in the plurality of oligonucleotides comprise (i) a random nucleotide sequence (B) at the 3' end having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B); and (ii) a unique sequence tag (X) at the 5' end; b) amplifying the tagged cDNA obtained in step (a) using a pair of primers, wherein the forward primer specifically anneals to a target sequence within the cDNA and wherein the reverse primer comprises a nucleic acid sequence that is at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X); and c) detecting and quantifying the nucleic acids amplified in step (b). Further provided herein is a plurality of oligonucleotides and kits comprising the same.

Description

[0001] PCT-Patent Application based on 24 199 354.2 Universitat Basel

[0002] Vossius Ref.: AJ2918 PCT BS

[0003] METHODS FOR SELECTIVELY QUANTIFYING RNA IN A BIOLOGICAL SAMPLE

[0004] BACKGROUND TO THE INVENTION

[0005] In biology and medicine, the current standard for nucleic acid quantification heavily relies on the in vitro amplification of said nucleic acid by the Polymerase Chain Reaction (PCR) method. PCR uses an enzyme from the class of template-dependent polymerases to exponentially amplify the target nucleic acid (RNA or DNA), under temperature and buffer conditions that are specific to each round of amplification. When a nucleic-acid-sensitive dye is used in such an exponentially-amplifying reaction, the quantity of target nucleic acid that builds up in the reaction can be tracked. A PCR reaction of this nature is called a quantitative polymerase chain reaction (qPCR)(Heid, Stevens et al. 1996).

[0006] The invention of qPCR (also known as real time PCR, or RT-PCR) in 1984 by Kary Mullis, rewarded with the 1993 Nobel Prize, had enormous consequences for the development of molecular biology. The most common application of qPCR is measuring the quantity of a specific RNA in a mixture of nucleic acids. This mixture can come from cell lysates, from blood samples, etc. The process involves an initial conversion of RNAs into complementary DNAs (cDNAs) using an enzyme called reverse transcriptase and then quantifying the cDNA that corresponds to the RNA of interest by qPCR. This entire process is called reverse transcriptase qPCR (RT-qPCR). In qPCR, the accumulation of fluorescent signal is indicative of the target nucleic acid being present in the initial mixture. The readout of a qPCR run is the Ct value, defined as the number of amplification cycles required for the signal to surpass the background (Smith and Osborn 2009). RT-qPCR is an extremely sensitive method that can identify RNAs whose abundance in the mixture is as low as a few in a hundred thousand molecules. This high sensitivity makes RT-qPCR the gold standard for diagnostics of viral infections, among others. However, the extremely high sensitivity makes the method prone to false positives, which are generated when DNAs and RNAs that are related to the target of interest are present in the initial sample. This is especially problematic when the initial mixture contains the DNA template of the RNA of interest. Current approaches to deal with DNA contamination are: 1) kits forthe specific extraction of RNA or 2) treatment of the sample with a DNA degrading enzyme (DNAse) prior to RT-qPCR, and 3) meticulous laboratory practices to avoid breaking open the nucleus and thus release the DNA during sample collection (Wan, Seth et al. 2010).

[0007] While these methods are reasonably effective in reducing DNA contamination, the precise efficiency of DNA removal from individual samples is unknown because the current practice does not include an assessment of this efficiency. This leads to undetermined fluctuations in the accuracy of RNA abundance estimates. Other drawbacks of the current approaches include a strong dependence on the quality of the RNA sample, expensive reagents, and technician time (Tavares, Alves et al. 2011).

[0008] Hence, there still exists a need in the art for improved PCR methods for selectively detecting and quantifying RNA targets in biological samples.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention is characterized in the herein provided embodiments and claims. In particular, the present invention relates, inter alia, to the following embodiments:

[0011] 1. A method for detecting and quantifying at least one target nucleic acid in a reverse transcriptase polymerase chain reaction (RT-PCR), the method comprising: a) reverse transcribing RNA present in a biological sample into complementary DNA (cDNA) using a plurality of oligonucleotides, wherein the oligonucleotides in the plurality of oligonucleotides comprise (i) a random nucleotide sequence (B) at the 3' end having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B); and

[0012] (ii) a unique sequence tag (X) at the 5' end; b) amplifying the tagged cDNA obtained in step (a) using a pair of primers, wherein the forward primer specifically anneals to a target sequence within the cDNA and wherein the reverse primer comprises a nucleic acid sequence that is at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X); and c) detecting and quantifying the nucleic acids amplified in step (b).

[0013] 2. The method of embodiment 1, wherein step (a) results in reverse transcription of the entire RNA in the biological sample into cDNA.

[0014] 3. The method of embodiment 1 or 2, wherein the method does not require a step of purifying the cDNA obtained in step (a) before the amplification in step (b).

[0015] 4. The method of any one of embodiments I to 3, wherein the detection and quantification of the nucleic acids amplified in step (b) comprises the use of a nucleic acid dye.

[0016] 5. The method of any one of embodiments I to 3, wherein the detection and quantification of the nucleic acids amplified in step (b) comprises the use of a nucleic acid probe comprising a detectable marker.

[0017] 6. The method of embodiment 5, wherein the nucleic acid probe is designed such that the detectable marker is released from the nucleic acid probe when the cDNA is amplified from the forward or reverse primer.

[0018] 7. The method of embodiment 5 or 6, wherein the detectable marker is a fluorescent marker, preferably wherein the nucleic acid probe further comprises a quencher that decreases the fluorescence intensity of the fluorescent marker when bound to the nucleic acid probe.

[0019] 8. The method of any one of embodiments 1 to 7, wherein two or more pairs of primers are added in step (b), preferably wherein the forward primers of the two or more primers specifically anneal to different target sequences within the cDNA and wherein the reverse primers of the two or more primers comprise a nucleic acid sequence that is at least partially identical to the nucleic acid sequence of the unique sequence tag.

[0020] 9. The method of embodiment 8, wherein the tagged cDNA obtained in step (a) is divided into two or more samples before the amplification step (b) and a pair of primers is added to each sample.

[0021] 10. A method for generating a plurality of tagged cDNA molecules, the method comprising: a) providing a biological sample comprising RNA; b) adding a plurality of oligonucleotides to the biological sample, wherein the oligonucleotides in the plurality of oligonucleotides comprise

[0022] (i) a random nucleotide sequence (B) at the 3' end having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B); and

[0023] (ii) a unique sequence tag (X) at the 5' end; and c) reverse transcribing the RNA in the biological sample into cDNA with a reverse transcriptase to obtain a plurality of tagged cDNA molecules.

[0024] 11. The method of any one of embodiments 1 to 10, wherein the oligonucleotides in the plurality of oligonucleotides have the format 5'-X-(L)-B-3', wherein X is the unique sequence tag, L is an optional linker, and B is the random nucleotide sequence.

[0025] 12. The method of embodiment 11, wherein the linker L is a nucleic acid linker. 13. The method of any one of embodiments 1 to 12, wherein the random nucleotide sequence (B) has a length of 4 to 9 nucleotides, preferably 5 to 7 nucleotides, most preferably 6 nucleotides.

[0026] 14. The method of any one of embodiments 1 to 13, wherein the plurality of oligonucleotides comprises at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000 or at least 4000 different random nucleotide sequences (B).

[0027] 15. The method of to any one of embodiments 1 to 14, wherein the plurality of oligonucleotides is a pool of random oligomers, wherein each random oligomer is linked to a unique sequence tag (X) at its 5' end.

[0028] 16. The method of any one of embodiments 1 to 15, wherein the unique sequence tag (X) is identical in all oligonucleotides comprised in the plurality of oligonucleotides.

[0029] 17. The method of any one of embodiments 1 to 16, wherein the sequence of the unique sequence tag (X) is absent in the organism from which the biological sample is derived.

[0030] 18. The method of any one of embodiments 1 to 17, wherein the unique sequence tag (X) has a length of 6 to 80 nucleotides, preferably 10 to 50 nucleotides, more preferably 15 to 30 nucleotides.

[0031] 19. The method of any one of embodiments 1 to 18, wherein the unique sequence tag (X) comprises a sequence selected from the group consisting of: a) 5'-TAG CCG TAA TCG TAG GAT CG-3' (SEQ ID NO: 1); b) 5'-CCG ATC ACC TTT AGA GCC AA-3' (SEQ ID NO: 2); c) 5'-CGC ACA TTA TTT TAG TGG GGT CAT GAG GTT AGG GAT GTT T-3' (SEQ ID NO: 3); d) 5'-GGG TGA TGC CCG TGC AAA TCA AAC TCT CGG AGA AGG CGA TAG GTT TTA CGG ATT TTT AGT-3' (SEQ ID NO: 4). 20. The method of any one of embodiments 1 to 19, wherein the biological sample is a purified nucleic acid sample, preferably wherein the purified nucleic acid sample comprises RNA or is suspected to comprise RNA.

[0032] 21. The method of embodiment 20, wherein the purified RNA sample is derived from the group consisting of: brain, spleen, bone, heart, vascular, lung, kidney, liver, intestine, muscle, blood, pituitary, endocrine glands, lymph node, epithelia, buccal mucosa, serum, plasma, cerebrospinal fluid, urine, saliva, biopsies, swabs and cytological specimens.

[0033] 22. A plurality of oligonucleotides having the format 5'-X-(L)-B-3', wherein X is a unique sequence tag, L is an optional linker, and B is a random nucleotide sequence having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B).

[0034] 23. The plurality of oligonucleotides of embodiment 22, wherein the random nucleotide sequence (B) has a length of 4 to 9 nucleotides, preferably 5 to 7 nucleotides, most preferably 6 nucleotides.

[0035] 24. The plurality of oligonucleotides of embodiment 22 or 23, wherein the plurality of oligonucleotides comprises at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000 or at least 4000 different random nucleotide sequences (B).

[0036] 25. The plurality of oligonucleotides of any one of embodiments 22 to 24, wherein the linker L is a nucleic acid linker.

[0037] 26. The plurality of oligonucleotides of any one of embodiments 22 to 25, wherein the unique sequence tag (X) is identical in all oligonucleotides comprised in the plurality of oligonucleotides. 27. The plurality of oligonucleotides of any one of embodiments 22 to 26, wherein the sequence of the unique sequence tag (X) is absent in a target organism.

[0038] 28. The plurality of oligonucleotides of any one of embodiments 22 to 27, wherein the unique sequence tag (X) has a length of 6 to 80 nucleotides, preferably 10 to 50 nucleotides, more preferably 15 to 30 nucleotides.

[0039] 29. The plurality of oligonucleotides of any one of embodiments 22 to 28, wherein the unique sequence tag (X) comprises a sequence selected from the group consisting of: a) 5'-TAG CCG TAA TCG TAG GAT CG-3' (SEQ ID NO: 1); b) 5'-CCG ATC ACC TTT AGA GCC AA-3' (SEQ ID NO: 2); c) 5'-CGC ACA TTA TTT TAG TGG GGT CAT GAG GTT AGG GAT GTT T-3' (SEQ ID NO: 3); d) 5'-GGG TGA TGC CCG TGC AAA TCA AAC TCT CGG AGA AGG CGA TAG GTT TTA CGG ATT TTT AGT-3' (SEQ ID NO: 4).

[0040] 30. A kit comprising the plurality of oligonucleotides of any one of embodiments 22 to 29.

[0041] 31. The kit of embodiment 30, further comprising one or more of: a reverse transcriptase enzyme, an RNAse H enzyme, a DNA polymerase, a buffer, at least one target-specific forward primer, at least one reverse primerthat is at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X), a nucleic acid dye, and / or a nucleic acid probe comprising a detectable marker.

[0042] Accordingly, in a particular embodiment, the invention relates to a method for detecting and quantifying at least one target nucleic acid in a reverse transcriptase polymerase chain reaction (RT-PCR), the method comprising: a) reverse transcribing RNA present in a biological sample into complementary DNA (cDNA) using a plurality of oligonucleotides, wherein the oligonucleotides in the plurality of oligonucleotides comprise (i) a random nucleotide sequence (B) at the 3' end having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B); and

[0043] (ii) a unique sequence tag (X) at the 5' end; b) amplifying the tagged cDNA obtained in step (a) using a pair of primers, wherein the forward primer specifically anneals to a target sequence within the cDNA and wherein the reverse primer comprises a nucleic acid sequence that is at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X); and c) detecting and quantifying the nucleic acids amplified in step (b).

[0044] That is, the present invention is directed to a method for detecting and quantifying a target nucleic acid, preferably a target RNA, in a biological sample, while avoiding unintentional amplification of genomic DNA (gDNA). This unintentional amplification of gDNA is a common problem in RNA detection via RT-PCR and often results in false positive results.

[0045] The inventors have surprisingly found that using a plurality of oligonucleotides comprising (i) a 3' random nucleotide sequence for hybridizing with RNA in the biological sample and (ii) a 5' unique nucleotide sequence tag produces a plurality of tagged cDNA molecules in a reverse transcription reaction. Subsequent PCR amplification with a combination of target and tagspecific primers enables exclusive amplification of the tagged cDNA, and avoiding the amplification of other DNAs in the sample, such as genomic DNA, thereby significantly lowering the risk of false positive results.

[0046] This method contrasts with previous approaches, such as those disclosed by Schuldiner et al. (Gene, 1990, 91(l):139-42) and WO 91 / 15601, where target-specific primers were employed both for the reverse transcription of RNA and the amplification of cDNA. These earlier methods carry a risk of amplifying residual genomic DNA present in the sample, as the targetspecific PCR primers can hybridize with both the cDNA and any contaminating DNA, leading to non-specific amplification and potentially confounding results. For example, it was shown in Fig. 1 A & B that the method according to the invention can be used to quantify specific RNAs in a sample. At the same time, quantification of these RNAs was not possible when the RT step was omitted, indicating that the tag-specific primer used in the PCR reaction cannot bind to residual DNA in the sample. When the same sample was analyzed with a commercial RT-PCR kit comprising traditional hexamer primers, i.e., without the unique sequence tag, and the obtained, non-tagged, cDNA was amplified with a targetspecific primer pair, quantification of the RNA was also possible. However, amplification with the target-specific primers could even be observed when the RT step was omitted. Since RNAs cannot serve as templates for DNA polymerases, this amplification observed in the absence of the RT step can only be explained by the presence of contaminating DNAs in the sample. This example demonstrates that the method according to the invention is insensitive to DNA contamination, contrary to commercial kits comprising untagged RT primers and targetspecific PCR primers, where contaminating DNA may be amplified with target-specific PCR primers to produce false positive results.

[0047] Furthermore, it was shown in Fig. 2 A & B that an additional step of DNA removal via DNAase treatment is crucial to avoid amplification of DNA contaminants when using a commercial kit. On the contrary, DNAse treatment was not required when using the method according to the invention.

[0048] Accordingly, one of the advantages of the method of the invention is that due to the tagging of the cDNA with a unique sequence tag and the specific amplification of tagged cDNA with a tag-specific primer, it is no longer necessary to remove or degrade DNA from the sample prior to the reverse transcription step. Thus, in certain embodiments, the invention relates to a method according to the invention, wherein a step of removing or degrading DNA in the sample prior to reverse transcription is no longer required.

[0049] DNA removal is a common step in RT-PCR and is performed prior to the RT reaction to avoid DNA contamination that can be amplified and produce false positive results. DNA removal is commonly achieved through degradation by addition of DNAse. The present invention is insensitive to DNA contamination because the plurality of oligonucleotides produces tagged cDNA during RT, ensuring that only RNA-derived cDNA molecules contain a unique sequence tag which is required for amplification of the cDNA.

[0050] To produce tagged cDNA molecules from RNA present in a sample, a plurality of oligonucleotides is designed to contain two functional sequences, a random nucleotide sequence (B) at the 3' end and a unique sequence tag (X) at the 5' end.

[0051] The random nucleotide sequence (B) hybridizes with a complementary or partially complementary RNA sequence in the sample and enables the initiation of the reverse transcription (RT) reaction.

[0052] The unique sequence tag (X) is unique in that it comprises a nucleotide sequence that is absent in the organism from which the sample is derived. During RT, said unique sequence tag (X) gets incorporated into cDNA as a 5' sequence tag. Amplification of the tagged cDNA by PCR can be initiated with a forward primer designed to hybridize with a target sequence in the tagged cDNA. The DNA strand obtained by amplification with the forward primer can then again be amplified with a reverse primer of identical or partially identical sequence as the unique sequence tag (X). This reverse primer hybridizes with a region in the DNA strand obtained by amplification with the forward primer that is complementary to the unique sequence tag (X) present in the cDNA molecule. By repeating these steps, a PCR reaction can be performed to amplify a target nucleotide sequence. Due to the absence of the unique target sequence (X) in the genomic DNA of the organism from which the sample is derived, amplification is selective for tagged cDNA. At the same time, amplification of contaminating DNA, such as genomic DNA or plasmid DNA, can be prevented due to the absence of an annealing site forthe reverse primer in the contaminating DNA. This process results in marked reduction of false positive results as shown in the appended Examples.

[0053] As explained herein, detecting and quantifying a target nucleic acid with the method according to the invention requires the use of a plurality of oligonucleotides in the reverse transcription step. The term "plurality of oligonucleotides" as used herein denotes a pool or group of oligonucleotides which can hybridize with RNA molecules to produce cDNA molecules in a reverse transcription reaction. That is, the "plurality of oligonucleotides" may also be referred to as a pool of reverse transcriptase primers or RT primers.

[0054] An "oligonucleotide" as used herein denotes linear oligomers of natural or modified nucleoside monomers linked by phosphodiester bonds or analogs thereof. Oligonucleotides include deoxyribonucleotides and ribonucleotides capable of specifically binding to a target. Usually, monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to several tens of monomeric units, e.g., 40-60. In certain embodiments, the oligonucleotides comprised in the plurality of oligonucleotides are DNA molecules.

[0055] Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5'-3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, "T" denotes deoxythymidine, and "U" denotes the ribonucleoside, uridine, unless otherwise noted.

[0056] The term "primer" as used herein refers to a short, single-stranded nucleic acid sequence, typically composed of DNA or RNA, that serves as a starting point for nucleic acid synthesis. In the context of reverse transcription (RT), primers are used to initiate the synthesis of complementary DNA (cDNA) from an RNA template by providing a free 3'-hydroxyl group to which nucleotides can be added by the reverse transcriptase enzyme. In PCR (Polymerase Chain Reaction), primers are designed to anneal to specific sequences within the cDNA or DNA template, enabling the DNA polymerase to extend the primer and synthesize new strands of DNA. Primers are essential for both RT and PCR processes, as they determine the specificity of the amplification by binding to complementary sequences in a target. The design of primers involves considerations such as length, melting temperature (Tm), and nucleotide composition to ensure efficient and specific hybridization under the chosen experimental conditions. As used herein, the terms "hybridization" and "annealing" are used interchangeably and refer to the base-pairing interaction of one oligonucleotide with another oligonucleotide (typically an antiparallel oligonucleotide) that results in formation of a duplex or other higher-ordered structure, typically termed a hybridization complex. The primary interaction between the antiparallel oligonucleotide molecules is typically base specific, e.g., A / T and G / C, by Watson / Crick and / or Hoogsteen-type hydrogen bonding. It is not a requirement that two oligonucleotides have 100% complementarity over their full length to achieve hybridization.

[0057] The oligonucleotides in the plurality of oligonucleotides comprise at least two functional fragments, a 5' unique sequence tag (X) and a 3' random nucleotide sequence (B). Whereas the random nucleotide sequence (B) is required for reverse transcribing RNA in the sample, the unique sequence tag (X) provides a unique hybridization site for the subsequent PCR reaction.

[0058] The term "complementary" means that one nucleic acid is identical to, or hybridizes selectively to, another nucleic acid molecule. Selectivity of hybridization exists when hybridization occurs that is more selective than total lack of specificity. Typically, selective hybridization will occur when there is at least about 55% identity over a stretch of at least 14- 25 nucleotides, preferably at least 65%, more preferably at least 75%, and most preferably at least 90%. Preferably, one nucleic acid hybridizes specifically to the other nucleic acid. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984).

[0059] The term "random nucleotide sequence" as used herein refers to a sequence of nucleotides that is designed to hybridize with a wide variety of RNA sequences present in the sample. This sequence typically ranges from 3 to 12 nucleotides in length and may include any combination of the four standard nucleotides (adenine, cytosine, guanine, and thymine / uracil). Accordingly, a "random nucleotide sequence" may be a nucleotide sequence with a length of 3 to 12 nucleotides, of which each position of the sequence can be occupied by one of the nucleotides A, T, C and / or G in a randomized order.

[0060] The randomness of the sequence allows for the initiation of reverse transcription across diverse RNA molecules, ensuring comprehensive cDNA synthesis from the total RNA present in the sample. The random nucleotide sequence (B) thus serves as a versatile primer binding site, facilitating the generation of tagged cDNA for subsequent amplification and analysis.

[0061] That is, the plurality of oligonucleotides used in the method of the invention comprises a plurality of random nucleotide sequences having lengths of 3 to 12 nucleotides, of which at least some will inevitably be complementary to RNA sequences in the biological sample. The hybridization of these sequences with the RNA produces priming sites for the initiation of the reverse transcription reaction. The random nucleotide sequence is preferably a DNA sequence and may bind to any type of RNA, including mRNA, miRNA, ncRNA, rRNA, and tRNA.

[0062] To allow for efficient reverse transcription of the RNA in the biological sample, it is required that the plurality of oligonucleotides comprises multiple random nucleotide sequences (B) that can hybridize with different parts of the RNA. That is, the plurality of oligonucleotides comprises at least two different random nucleotide sequences that may serve as priming sites fora reverse transcription reaction. In certain embodiments, the invention relates to a method according to the invention, wherein the plurality of oligonucleotides comprises at least 2, least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 12,000, at least 14,000, at least 16,000, at least 18,000, at least 20,000, at least 40,000, at least 60,000, at least 80,000, at least 100,000, at least 500,000, at least 1,000,000, at or least 1,500,000 different random nucleotide sequences (B).

[0063] It is preferred herein, that the number of different random nucleotide sequences in the plurality of oligonucleotides needs to be sufficiently high to allow reverse transcription of all, or at least most, of the RNA in a sample. The skilled person would understand that the number of different random nucleotide sequences that are required to achieve complete or nearly complete reverse transcription of an RNA sample depends on the length of the random nucleotide sequences. That is, shorter random nucleotide sequences will generally hybridize to more RNA molecules than longer random nucleotide sequences. It is thus to be expected that the number of different random nucleotide sequences that is required to achieve complete or nearly complete reverse transcription of an RNA sample increases with the length of the random nucleotide sequences (B).

[0064] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the plurality of oligonucleotides comprises a plurality of different random nucleotide sequences (B) that is sufficient to achieve reverse transcription of at least 50%, of at least 60%, of at least 70%, of at least 80%, of at least 90%, of at least 95%, or of at least 99% of the RNA in the biological sample into cDNA.

[0065] The skilled person is capable of selecting a suitable length and diversity of the random nucleotide sequence (B) that allows for sufficient reverse transcription of RNA from the biological sample into cDNA. Furthermore, the skilled person can determine the efficiency of the reverse transcription process. For example, by direct sequencing of total RNA and sequencing of the cDNA separately, one can compare the two datasets to calculate the degree of reverse transcription. This method provides detailed insights into the efficiency and completeness of the reverse transcription process, allowing for a comprehensive evaluation of how effectively the RNA has been converted into cDNA.

[0066] The random nucleotide sequences (B) comprised in the in the plurality of oligonucleotides may have a length of 3 to 12 nucleotides. In certain embodiments, all random nucleotide sequences (B) comprised in the in the plurality of oligonucleotides have the same length. In such embodiments, all random nucleotide sequences (B) comprised in the plurality of nucleotides may have a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides.

[0067] In certain embodiments, the random nucleotide sequences (B) comprised in the in the plurality of oligonucleotides may have different lengths. In such embodiments, the random nucleotide sequences (B) comprised in the plurality of nucleotides may have an average length of 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides.

[0068] In certain embodiments, the random nucleotide sequences (B) comprised in the plurality of nucleotides may have a(n average) length of 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 12, 8 to 11, 8 to 10, 9 to 12, 9 to 11, or 10 to 12 nucleotides.

[0069] In a particular embodiment, the invention relates to the method according to the invention, wherein the random nucleotide sequence (B) has a length of 4 to 9 nucleotides, preferably 5 to 7 nucleotides, most preferably 6 nucleotides.

[0070] That is, in certain embodiments, the random nucleotide sequence (B) comprised in the plurality of oligonucleotides may be a random pentamer. In such embodiments, the plurality of oligonucleotides may comprise at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, preferably at least 800, more preferably at least 900, most preferably at least 1000, different random nucleotide sequences (B).

[0071] In a preferred embodiment, the random nucleotide sequence (B) comprised in the plurality of oligonucleotides may be a random hexamer. In such embodiments, the plurality of oligonucleotides may comprise at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, preferably at least 2000, more preferably at least 3000, and most preferably at least 4000 different random nucleotide sequences (B).

[0072] In another embodiment, the random nucleotide sequence (B) comprised in the plurality of oligonucleotides may be a random heptamer In such embodiments, the plurality of oligonucleotides may comprise at least 10, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10000, preferably at least 12000, more preferably at least 14000, and most preferably at least 16000 different random nucleotide sequences (B).

[0073] In another embodiment, the random nucleotide sequence (B) comprised in the plurality of oligonucleotides may be a mix of random pentamers, hexamer and / or heptamers. In such embodiments, the plurality of oligonucleotides may comprise at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10000, preferably at least 12000, more preferably at least 14000, and most preferably at least 16000 different random nucleotide sequences (B).

[0074] In certain embodiments, the plurality of oligonucleotides comprises all possible random oligomers of a given length. In such embodiments, the number of possible random nucleotide sequences (B) in the plurality of oligonucleotides is dependent on the length of the random nucleotide sequence (B). For example, a plurality of oligonucleotides with a random nucleotide sequence (B) length of 3 can comprise a maximal number of 43different nucleotides and a plurality of oligonucleotides with a random nucleotide sequence (B) length of 12 can comprise a maximal number of 412different nucleotides.

[0075] Thus, in certain embodiments, the invention relates to a method according to the invention, wherein the plurality of oligonucleotides comprises 43, 44, 45, 46, 47, 48, 49, 410, 411, or 412different random nucleotide sequences (B).

[0076] In certain embodiments, the random nucleotide sequence (B) comprised in the plurality of oligonucleotides may be a random hexamer and the plurality of oligonucleotides may comprise 46or 4096 different random nucleotide sequences (B).

[0077] In all embodiments disclosed herein, it is preferred that each oligonucleotide comprised in the plurality of oligonucleotides comprises a single random nucleotide sequence (B), more preferably at the 3' end of the oligonucleotide.

[0078] The diversity in the plurality of oligonucleotides mainly results from the different random nucleotide sequences (B) at the 3' end of the oligonucleotides, while the unique sequence tag (X) located at the 5' end of the oligonucleotides is less diverse or may even be identical in all oligonucleotides comprised in the plurality of oligonucleotides.

[0079] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the plurality of oligonucleotides is a pool of random nucleotide sequences (B), wherein each random nucleotide sequences (B) is linked to a unique sequence tag (X) at its 5' end.

[0080] The term "unique sequence tag" as used herein denotes a nucleotide sequence that is deliberately designed to be absent from the genome of the organism from which the sample was derived and thus cannot hybridize with residual DNA in the biological sample, such as genomic DNA (gDNA) of the organism from which the sample was derived. In certain embodiments, the unique sequence tag may be a nucleotide sequence that does not anneal to DNA in the biological sample, preferably at a temperature of 23 degrees centigrade or higher, more preferably at a temperature of 30 degrees centigrade or higher, even more preferably at a temperature of 40 degrees centigrade or higher, most preferably at a temperature of 50 degrees centigrade or higher.

[0081] To determine whether the unique sequence tag does not anneal at these temperatures, hybridization assays or melting curve analyses can be performed. In hybridization assays, the unique sequence tag is labeled and mixed with the DNA sample, and the degree of hybridization is monitored at increasing temperatures. In melting curve analysis, the mixture is gradually heated, and the dissociation of DNA duplexes is observed. Both methods provide empirical evidence to confirm that the unique sequence tag does not hybridize with the DNA at the specified temperatures, ensuring the specificity and reliability of the amplification process. The skilled person is aware of such method and capable of designing the appropriate experiments.

[0082] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the sequence of the unique sequence tag (X) is absent in the organism from which the biological sample is derived.

[0083] The unique sequence tag (X) may have any suitable length. Preferably, the unique sequence tag (X) has a length that is sufficient to enable hybridization of a PCR primer in the subsequent PCR reaction under standard PCR conditions. That is, in certain embodiments, the invention relates to the method according to the invention, wherein the unique sequence tag (X) has a length of 6 to 80 nucleotides, preferably 10 to 50 nucleotides, more preferably 15 to 30 nucleotides.

[0084] In certain embodiments, the unique sequence tag (X) may have a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.

[0085] In certain embodiments, the unique sequence tag (X) may have a length of: 6 to 10, 6 to 15, 6 to 20, 6 to 25, 6 to 30, 6 to 35, 6 to 40, 6 to 45, 6 to 50, 6 to 55, 6 to 60, 6 to 65, 6 to 70, 6 to 75, 6 to 80, 10 to 15, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 80, 30 to 40, 30 to 50, 30 to 60, 30 to 70, 30 to 80, 40 to 50, 40 to 60, 40 to 70, 40 to 80, 50 to 60, 50 to 70, 50 to 80, 60 to 70, 60 to 80, or 70 to 80 nucleotides.

[0086] In certain embodiments, the biological sample used in the method of the invention has been obtained from a human and the unique sequence tag (X) is a nucleotide sequence that is absent in the human genome. Exemplary sequence that may be used as unique sequence tag (X) when analyzing human-derived samples include the sequences set forth in SEQ ID NO:l-4.

[0087] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the unique sequence tag (X) comprises a sequence selected from the group consisting of: a) 5'-TAG CCG TAA TCG TAG GAT CG-3' (SEQ ID NO: 1); b) 5'-CCG ATC ACC TTT AGA GCC AA-3' (SEQ ID NO: 2); c) 5'-CGC ACA TTA TTT TAG TGG GGT CAT GAG GTT AGG GAT GTT T-3' (SEQ ID NO: 3); d) 5'-GGG TGA TGC CCG TGC AAA TCA AAC TCT CGG AGA AGG CGA TAG GTT TTA CGG ATT TTT AGT-3' (SEQ ID NO: 4).

[0088] Bioinformatic tools for predicting nucleotide sequences that are absent in the genomic DNA of an organism of interest, in particular a human, are known in the art and may be used for generating the unique sequence tag (X) comprised in the plurality of oligonucleotides. Nonlimiting examples include the Tagenerator tool developed by Pinto et al. (BMC Biotechnol. 2006; 6: 31).

[0089] The oligonucleotides comprised in the plurality of oligonucleotides may comprise one or more unique sequence tags (X). That is, all oligonucleotides comprised in the plurality of oligonucleotides may comprise the same unique sequence tag (X). Using a plurality of oligonucleotides comprising the same unique sequence tag (X) in the method of the invention offers the advantage that the same reverse primer may be used for the amplification of different target sequences. That is, the same reverse primer may be combined with different target-specific forward primers.

[0090] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the unique sequence tag (X) is identical in all oligonucleotides comprised in the plurality of oligonucleotides.

[0091] Alternatively, two or more unique sequence tags (X) may be comprised in the plurality of oligonucleotides. That is, in certain embodiments, the plurality of oligonucleotides may comprise 2, 3, 4, 5, 6 or more unique sequence tags (X). The different unique sequence tags (X) comprised in the plurality of oligonucleotides may be partially overlapping or may be entirely different.

[0092] The unique sequence tag (X) may be linked to the 5' end of the random nucleotide sequence (B) with an optional linker sequence. That is, in a particular embodiment, the invention relates to the method according to the invention, wherein the oligonucleotides in the plurality of oligonucleotides have the format 5'-X-(L)-B-3', wherein X is the unique sequence tag, L is an optional linker, and B is the random nucleotide sequence.

[0093] Preferably, the linker L is a nucleic acid linker that connects the unique sequence tag (X) with the random nucleotide sequence (B). The term "nucleic acid linker" or "linker sequence" or "linker" as used herein denotes an optional oligonucleotide sequence which connects the random nucleotide sequence (B) with the unique sequence tag (X).

[0094] The linker is preferably a nucleic acid linker, more preferably a DNA linker.

[0095] In certain embodiments, the linker has a length of 1 to 20 nucleotides. In certain embodiments, the linker has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 1-5, 6-10, 11-15, 16-20, 1-10 or 11-20 nucleotides.

[0096] The linker may have any nucleotide sequence. However, it is preferred that the linker has a nucleotide sequence which does not hybridize with target RNA sequences or genomic DNA sequences in the biological sample to reduce the risk of false positive results.

[0097] The plurality of oligonucleotides described herein above is used in the method of the invention for the reverse transcription of RNA in a biological sample, preferably for the reverse transcription of the entire RNA in a biological sample.

[0098] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein reverse transcription with the plurality of oligonucleotides results in reverse transcription of the entire RNA in the biological sample into cDNA.

[0099] The term "entire RNA" or "total RNA" as used herein denotes all RNA molecules that are present in a biological sample, including all RNA species (such as mRNA, miRNA, ncRNA, rRNA, and tRNA and other non-coding RNAs). In the context of reverse transcription, transcription of the entire RNA in a biological sample to cDNA may comprise values above 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%. RT efficiency may depend on several factors such as RNA quality and degree of degradation, RT enzyme quality and type of enzyme, RNA secondary structures and primer design. Thus, reverse transcription of 100% of the RNA in a biological sample may not be technically feasible. In a preferred embodiment, reverse transcription with the plurality of oligonucleotides results in reverse transcription of at least 90%, at least 95%, at least 98, at least 99%, at least 99.5% or at least 99.9% of the RNA in the biological sample. The term "reverse transcription" ("RT") as used herein denotes a transcription reaction mediated by the reverse transcriptase enzyme in which RNA is transcribed into cDNA.

[0100] Some reverse transcriptases may comprise non-template terminal transferase activity. When a reverse transcriptase comprising non-template terminal transferase activity reaches the end of a template, it can add three or more non-template residues, such as three or more non- template cytosine residues. It is thus preferable that the reverse transcriptase used in the method of the present invention lacks or has reduced non-template terminal transferase activity. The reverse transcriptase used in the present invention may be engineered to have reduced non-template terminal transferase activity.

[0101] In certain embodiments, Superscript IV™ reverse transcriptase is used for this purpose. In certain embodiments, Maxima™ reverse transcriptase is used for this purpose. In certain embodiments, Protoscript II™ reverse transcriptase is used for this purpose. In certain embodiments, Maloney murine leukemia virus reverse transcriptase (MMLV-RT) is used for this purpose. In certain embodiments, HighScriber™ Reverse Transcriptase is used for this purpose. In certain embodiments, a terminal deoxynucleotidyl transferase is used for this purpose. In certain embodiments, avian myeloblastosis virus (AMV) reverse transcriptase is used for this purpose.

[0102] Reverse transcription is performed to generate a cDNA sample, wherein each cDNA molecule in the cDNA sample comprises a unique sequence tag (X) at the 5' end.

[0103] Accordingly, in a particular embodiment, the invention relates to a method for generating a plurality of tagged cDNA molecules comprising: a) providing a biological sample comprising RNA; b) adding a plurality of oligonucleotides to the biological sample, wherein the oligonucleotides in the plurality of oligonucleotides comprise

[0104] (i) a random nucleotide sequence (B) at the 3' end having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B); and (ii) a unique sequence tag (X) at the 5' end; and c) reverse transcribing the RNA in the biological sample into cDNA with a reverse transcriptase to obtain a plurality of tagged cDNA molecules.

[0105] That is, in one aspect, the invention relates to the reverse transcription step alone, i.e., independent of a subsequent PCR amplification step.

[0106] However, the cDNA obtained with the method according to the invention is preferably used as template in a subsequent PCR reaction.

[0107] Thus, in certain embodiments, the tagged cDNA, obtained in a reverse transcription reaction with the plurality of oligonucleotides, is amplified in a PCR reaction using a pair of primers.

[0108] Current methods for RNA-derived cDNA amplification require a cDNA purification step to remove reverse transcription primers from the sample. This step is crucial to prevent the reverse transcription primers from hybridizing with residual genomic DNA during the amplification process, which can result in non-specific amplification and potentially misleading results. The present invention comprises a pool of oligonucleotides that produce tagged cDNA from total RNA. As only tagged cDNA molecules are amplified in the subsequent PCR due to the choice of PCR primers, the method of the present invention is insensitive to genomic DNA contamination. Consequently, removal of the plurality of oligonucleotides prior to PCR amplification is not required in the method of the invention.

[0109] Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the method does not require a step of purifying the cDNA obtained by reverse transcription with the plurality of oligonucleotides before the PCR amplification step. That is, in certain embodiments, the PCR primers are added directly to the cDNA sample obtained in the reverse transcription reaction.

[0110] The term "Polymerase chain reaction" or "PCR" as used herein denotes a method whereby a specific segment of a target double-stranded DNA is amplified in a geometric progression. PCR is well known to those of skill in the art; see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990. The terms "real time PCR", "reverse transcription PCR" or "quantitative PCR" ("RT-PCR", "qPCR" or "RT- qPCR") as used herein denotes to real-time detection and quantitation of amplicon generated in a PCR. Detection and quantification may proceed through use of various detection agents, many of which are described herein.

[0111] The terms "amplification" and "amplifying" refer generally to any process that results in an increase in the copy number of a molecule or set of related molecules. Components of an amplification reaction may include, but are not limited to, e.g., primers, a polynucleotide template, nucleic acid polymerase, nucleotides, dNTPs and the like. The term amplifying typically refers to an exponential increase in target sequence. Amplification typically starts from a small amount of a template (e.g. a single copy of a template), where the amplified material is typically detectable. Amplification of the template encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a template may be effected by a polymerase chain reaction (PCR).

[0112] Preferably, the pair of primers used in the PCR reaction comprises a forward primer that specifically anneals to a target sequence within the cDNA and a reverse primer that is at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X).

[0113] That is, the forward primer used in the PCR step is target specific. The skilled person is capable of designing primers that specifically anneal to a target sequence in a cDNA. For example, bioinformatic tools may be used to identify a target sequence in a cDNA and / or to generate a primer sequence that specifically anneals to this target sequence. Typically, the design of the forward primer involves selecting a sequence that is complementary to a region within the target sequence, ensuring specific binding and initiation of the PCR amplification.

[0114] The term "target sequence" as used herein refers to a specific nucleotide sequence within the cDNA that is of interest for amplification and subsequent analysis. This sequence is typically derived from a gene or a region of a genome that is being studied, and it is selected based on its relevance to the biological or diagnostic objective of the experiment. The target sequence within a cDNA typically reflects the sequence of DNA that is transcribed to RNA. Where the DNA sequence that is transcribed to RNA is a gene, the target sequence within the cDNA typically reflects the coding sequence of the gene, but lacks introns and regulatory sequences.

[0115] It is to be understood that within the present invention, the "target nucleic acid" is the RNA of interest in the biological sample, and the "target sequence within the cDNA" is the specific region of the cDNA that corresponds to a part of this RNA. The forward primer binds to this target sequence within the cDNA to ensure specific amplification of the cDNA derived from the target nucleic acid.

[0116] The reverse primer, on the other hand, is designed to be at least partially identical to the unique sequence tag (X) that was incorporated during the reverse transcription reaction. This design ensures that only those cDNA molecules that have been tagged with the unique sequence tag are amplified, thereby increasing the specificity and reducing the likelihood of amplifying contaminating DNA in the sample.

[0117] In a preferred embodiment, the reverse primer is identical to the nucleic acid sequence of the unique sequence tag (X). However, it is also sufficient if the reverse primer is partially identical to the nucleic acid sequence of the unique sequence tag (X). For example, the unique sequence tag (X) may be longer than the reverse primer, resulting in only partial identity.

[0118] In certain embodiments, the reverse primer sequence is sufficiently identical to the unique sequence tag (X) to enable hybridization. Preferably, the reverse primer is at least about 55% identical, more preferably at least 65% identical, even more preferably at least 75% identical, and most preferably at least 90% identical to the unique sequence tag (X).

[0119] Preferably, the reverse primer and the unique sequence tag (X) share at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides that are identical in sequence, or differ only in 1, 2 or 3 positions. These contiguous nucleotides are preferably located at the 3' end of the reverse primer to ensure specific binding and efficient initiation of PCR amplification. More preferably, the entire sequence of the reverse primer is fully contained within the sequence of the unique sequence tag (X). In certain embodiments, the sequence of the reverse primer and the unique sequence tag (X) may differ in 1, 2, or 3 positions, as long as the reverse primer can still initiate specific amplification of the target sequence. These minor differences should not significantly affect the primer's ability to initiate PCR amplification.

[0120] In one embodiment, a single primer pair may be used to amplify and / or detect a target sequence within a cDNA molecule. As explained above, the primer pair may consist of a targetspecific forward primer that specifically hybridizes with a target sequence within the cDNA and a reverse primer that is at least partially identical to the sequence of the unique sequence tag (X).

[0121] However, the method of the invention is also suitable for multiplexing the detection and quantification of multiple target nucleic acids. This is possible due to the use of a plurality of oligonucleotides comprising random nucleotide sequences (B) in the reverse transcription step, which will result in reverse transcription of the entire RNA in a biological sample, or at least a substantial part thereof. By reverse transcribing the entire RNA into cDNA, it is possible to amplify multiple target sequences within the cDNA, even if these cDNA molecules were derived from different RNA molecules. This multiplexing approach enhances the versatility and efficiency of the method, allowing for comprehensive analysis of multiple targets in a single reaction.

[0122] For example, it has been demonstrated in Example 3, that the method of the invention can be used to quantify two different target sequences when starting from the same cDNA sample.

[0123] This is, in contrast to previous methods, as the one disclosed in WO 91 / 15601, where only a specific region of a target RNA was reverse transcribed and subsequently amplified with target-specific primers.

[0124] Thus, in a particular embodiment, the invention relates to the method of the invention, wherein two or more pairs of primers are added in step (b), preferably wherein the forward primers of the two or more primers specifically anneal to different target sequences within the cDNA and wherein the reverse primers of the two or more primers comprise a nucleic acid sequence that is at least partially identical to the nucleic acid sequence of the unique sequence tag.

[0125] In certain embodiments, the amplification of tagged cDNA in a PCR reaction is performed with one, two, three, four or more pairs of primers, preferably wherein the forward primers are designed to specifically anneal to at least one target sequence and the reverse primers are at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X). That is, different forward primers may be combined with the same reverse primer. The different forward primers hybridize with different target sequences within the cDNA. These different target sequences may be associated with the same target nucleic acid in the sample or with different target nucleic acids in the sample.

[0126] In certain embodiments, multiple target sequences are amplified simultaneously in the same reaction using two or more target specific forward primers and at least one reverse primer that is at least partially identical to the unique sequence tag (X). In certain embodiments, two or more reverse primers of varying sequence that are at least partially identical to one or more unique sequence tag (X) may be used.

[0127] In certain embodiments, the oligonucleotides in the plurality of oligonucleotides may be used as reverse primers for the amplification of tagged cDNA in a PCR reaction. In such embodiments, only one or more target-specific forward primers need to be added to initiate the PCR reaction.

[0128] Within the present invention, two or more pairs of primers, preferably wherein the two or more pairs of primers share a common reverse primer, may be added simultaneously to the same sample.

[0129] However, it is preferred herein that when multiple primer pairs are used in the PCR reaction, the sample is divided into multiple aliquots and each aliquot is analyzed with a single pair of primers. In certain embodiments, a biological sample comprising RNA may be divided into multiple aliquots. Each aliquot may then be reverse transcribed into cDNA using the plurality of oligonucleotides described herein. Subsequently, each cDNA aliquot may be mixed with a different pair of primers, wherein each pair of primers preferably comprises a distinct targetspecific forward primer and a reverse primer that is at least partially identical to the nucleic acid sequence of the unique sequence tag (X). This approach allows for the simultaneous amplification of multiple target sequences from the same biological sample.

[0130] Alternatively, the RNA in the biological sample may first be reverse transcribed into cDNA and the obtained cDNA may then be divided into multiple aliquots. Subsequently, each cDNA aliquot may be mixed with a different pair of primers, wherein each pair of primers preferably comprises a distinct target-specific forward primer and a reverse primer that is at least partially identical to the nucleic acid sequence of the unique sequence tag (X).

[0131] Accordingly, in a particular embodiment, the invention relates to the method of the invention, wherein the tagged cDNA obtained in step (a) is divided into two or more samples before the amplification step (b) and a pair of primers is added to each sample.

[0132] In certain embodiments, the invention relates to the method according to the invention, wherein the biological sample comprising RNA or the tagged cDNA obtained by reverse transcription is divided into 2, 3, 4, 5, 6, 7, 8, 9, 10 or more samples before the PCR amplification.

[0133] In certain embodiments, the invention relates to the method according to the invention, wherein the biological sample comprising RNA or the tagged cDNA obtained by reverse transcription is divided into 2-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91- 100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, 8000- 9000, 9000-10000 or more samples before the PCR amplification.

[0134] In certain embodiments, the invention relates to a method according to the invention, wherein at least one pair of primers is added to each cDNA aliquot to initiate PCR amplification, comprising forward primers which specifically anneal to different target sequences within the cDNA and reverse primers that are identical or at least partially identical to the nucleic acid sequence of the unique sequence tag (X).

[0135] In a preferred embodiment, the invention relates to the method according to the invention, wherein one specific primer pair is added to each cDNA aliquot to initiate the PCR amplification, comprising one target-specific forward primer and one reverse primer that is identical or at least partially identical to the nucleic acid sequence of the unique sequence tag (X).

[0136] In certain embodiments, the invention relates to the method according to the invention, wherein two or more target-specific forward primers and one reverse primer that is identical or at least partially identical to the nucleic acid sequence of the unique sequence tag (X) are added to each cDNA aliquot to initiate the PCR reaction.

[0137] In certain embodiments, the invention relates to the method according to the invention, wherein two or more target-specific forward primers and two or more reverse primers that are identical or at least partially identical to the nucleic acid sequence of the unique sequence tag (X) are added to each cDNA aliquot to initiate the PCR reaction.

[0138] The skilled person is capable of selecting the length and sequence of primers for use in PCR. The PCR reaction can be performed based on principles and procedures known to the skilled person. That is, the skilled person is capable of identifying suitable PCR conditions to achieve sufficient amplification of a target sequence. The PCR amplification conditions, including annealing temperature, extension time, and cycle number, may be optimized by the skilled person to achieve efficient and specific amplification of the tagged cDNA. For example, the annealing temperature can be adjusted based on the melting temperatures (Tm) of the primers, and the extension time can be set according to the expected length of the target cDNA.

[0139] The nucleic acids amplified in the PCR step are subsequently detected and quantified. The term "detection", as used herein, refers to the process of identifying the presence of specific nucleic acid sequences in a sample. Amplified nucleic acids may be detected using any method known in the art. For example, detection methods may include gel electrophoresis followed by staining with ethidium bromide.

[0140] However, it is preferred herein that the amplified nucleic acids are quantified. The term "quantification," as used herein, refers to the process of determining the amount or concentration of specific nucleic acid sequences in a sample. Quantification can be achieved through techniques such as quantitative PCR (qPCR), which utilizes fluorescent dyes or probes to measure the amount of amplified product in real-time, providing both qualitative and quantitative data. Additionally, digital PCR (dPCR) can be employed for absolute quantification of nucleic acids, offering high sensitivity and precision by partitioning the sample into many individual reactions. These quantification methods enable precise and accurate measurement of nucleic acid levels, facilitating a wide range of applications in research, diagnostics, and biotechnology.

[0141] Preferably, amplified nucleic acids are quantified in real time, meaning that the quantification occurs concurrently with the amplification process. Real-time quantification offers several advantages, including the ability to monitor the progress of the reaction as it happens, providing immediate feedback on the efficiency and specificity of the amplification.

[0142] In a particular embodiment, the invention relates to the method according to the invention, wherein the detection and quantification of the amplified nucleic acids comprises the use of a nucleic acid dye.

[0143] Nucleic acid dyes encompass both "intercalating agents" and "non-intercalating agents". An "intercalating agent" is an agent or moiety capable of non-covalent insertion between stacked base pairs in the nucleic acid double helix. Intercalating agents, such as ethidium bromide, fluoresce more intensely when intercalated into double-stranded DNA than when bound to single-stranded DNA, RNA, or in solution. Other intercalating agents, such as actinomycin D, exhibit a change in the fluorescence spectra when bound to double-stranded DNA. Any intercalating agent that provides a detectable signal that is distinguishable when the agent is bound to double-stranded DNA or unbound is suitable for use with the invention described herein. "Non-intercalating agents" are also suitable. For example, Hoechst 33258 (Searle & Embrey, 1990, Nuc. Acids Res. 18(13):3753-3762) exhibits altered fluorescence with increasing amount of target. Hoechst 33258 is a member of a class of DNA-binding compounds commonly referred to as "groove binders". These compounds recognize and bind the minor groove of duplex DNA.

[0144] Examples of nucleic acid dyes, inclusive of both intercalating agents and non-intercalating agents, include but are not limited to ethidium bromide, SYBR™ Green, PICOGREEN®, SYBR™ Gold, SYTO® 9, SYTO® 13, SYTO® 16, SYTOX® blue, chromomycin A3, Os[(bpy)2DPPZ]2+, BEBO, BOXTO, EVAGREEN®, propidium iodide, chromomycin, mithramycin, thiazole orange, CYTRAK ORANGE™, LDS 751, 7-AAD, SYTOX® green, SYTOX® orange, TOTO-3, DRAG5, DRAG7, ResoLight, acridine orange, Hoechst 33258, TOTO-1, YOYO-1, YO-PRO-1, TO-PRO-3, and 4', 6- diamidino-2-phenylindole ("DAPI").

[0145] In a preferred embodiment, amplified nucleic acids are quantified using an intercalating fluorescent dye, such as SYBR™ Green.

[0146] Alternatively, amplified nucleic acids may be quantified using a nucleic acid probe comprising a detectable marker. Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the detection and quantification of the amplified nucleic acids comprises the use of a nucleic acid probe comprising a detectable marker.

[0147] The principle of nucleic acid probes in qPCR involves the use of a sequence-specific probe that hybridizes to a target sequence during the PCR amplification process. This probe is labeled with a detectable marker, such as a fluorescent dye, at one end and a quencher molecule at the other end. When the probe is intact, the proximity of the quencher to the detectable marker suppresses the detectable signal. During PCR amplification, the DNA polymerase cleaves the probe, separating the detectable marker from the quencher, resulting in an increase in detectable signal. The signal intensity may be measured in real-time and is directly proportional to the amount of amplified target DNA, allowing for precise quantification. Accordingly, in a particular embodiment, the invention relates to a method according to the invention, wherein the nucleic acid probe is designed such that the detectable marker is released from the nucleic acid probe when the cDNA is amplified from the forward or reverse primer.

[0148] The "detectable marker" comprised in the nucleic acid probe is preferably a fluorescent molecule, such as FAM, TET, HEX, VIC, Cy3, Cy5, fluorescein, rhodamine, Oregon Green, eosin, Texas Red, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, R-phycoerythrin, peridinin chlorophyll protein, FluorX, BODIPY- fluorescein, Cy2, Cy3B, Cy3.5, Cy5.5, Cy7, TRITC, lissamine rhodamine B, or allophycocyanin.

[0149] The term "nucleic acid probe" refers to a nucleic acid molecule used for detecting amplification of a target sequence, typically through a mechanism involving 5' to 3' nuclease activity. The term "5' to 3' nuclease activity" or "5'-3' nuclease activity" refers to the activity of a nucleic acid polymerase, whereby nucleotides are removed from the 5' end of a nucleic acid strand during nucleic acid strand synthesis. Some enzymes that exhibit 5' to 3' nuclease activity are known as 5' to 3' exonucleases. Furthermore, the detection of a target nucleic acid utilizing the 5' to 3' nuclease activity can be performed using a "TAQMAN®" or "5'-nuclease assay," as described in U.S. Patent Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al., 1988, Proc. Natl. Acad. Sci. USA 88: 7276-7280. In the TaqMan® assay, labeled detection probes that hybridize within the amplified region are present during the amplification reaction. These probes are modified to prevent them from acting as primers for DNA synthesis. The amplification is performed using a DNA polymerase with 5' to 3' exonuclease activity. During each synthesis step of the amplification, any probe that hybridizes to the target sequence downstream from the primer being extended is degraded by the 5' to 3' exonuclease activity of the DNA polymerase. Thus, the synthesis of a new target strand also results in the degradation of a probe, and the accumulation of the degradation product provides a measure of the synthesis of target sequences.

[0150] Any method suitable for detecting degradation products can be used in a 5' nuclease assay.

[0151] Often, the nucleic acid probe is labeled with two fluorescent dyes: a reporter dye and a quencher dye. The reporter dye is typically attached to the 5' terminus of the probe, while the quencher dye is attached to an internal site or the 3' terminus. In the unhybridized state, the proximity of the quencher dye suppresses the fluorescence of the reporter dye. During amplification, the 5' to 3' exonuclease activity of the DNA polymerase cleaves the probe between the two dyes, separating the quencher from the reporter dye. This cleavage results in the elimination of quenching and an increase in fluorescence from the reporter dye. The accumulation of degradation products is monitored by measuring the increase in fluorescence. U.S. Patent Nos. 5,491,063 and 5,571,673 describe alternative methods for detecting probe degradation that occurs concomitant with amplification.

[0152] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the detectable marker is a fluorescent marker, preferably wherein the nucleic acid probe further comprises a quencher that decreases the fluorescence intensity of the fluorescent marker when bound to the nucleic acid probe.

[0153] A 5' nuclease assay for the detection and quantification of a target nucleic acid can employ any polymerase that has 5' to 3' exonuclease activity. In some instances, the polymerases with 5' nuclease activity are thermostable and thermoactive nucleic acid polymerases. Such thermostable polymerases include, but are not limited to, native and recombinant forms of polymerases from various species of the eubacterial genera Thermus, Thermotoga, and Thermosipho, as well as chimeric forms thereof. For example, Thermus species polymerases that can be used include Thermus aquaticus (Taq) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus species Z05 (Z05) DNA polymerase, Thermus species spsl7 (spsl7), and Thermus species Z05, as described in U.S. Patent Nos. 5,405,774; 5,352,600; 5,079,352; 4,889,818; 5,466,591; 5,618,711; 5,674,738; and 5,795,762.

[0154] The skilled person is capable of designing a nucleic acid probe that allows for quantifying the amplification of a target sequence. This involves selecting appropriate sequences that are complementary to the target sequence in the cDNA and incorporating detectable markers, such as fluorescent dyes, to facilitate real-time monitoring. The design process includes optimizing the probe's length, melting temperature, and specificity to ensure efficient hybridization and accurate detection. Additionally, the skilled person can utilize bioinformatics tools to identify target regions and predict probe performance, ensuring that the probe will function effectively under the chosen experimental conditions.

[0155] The method of the present invention is performed on a biological sample that contains a target nucleic acid or is suspected to contain a target nucleic acid. Preferably, the target nucleic acid is an RNA. Thus, the biological sample is preferably a sample that contains RNA or is suspected to contain RNA.

[0156] The term "biological sample" as used herein denotes a biological specimen or culture (e.g., microbiological cultures) that comprises or is suspected to comprise nucleic acids and / or target nucleic acids.

[0157] A "biological sample" may include, but is not limited to, amniotic fluid, biopsy sample, blood, bone, brain, breast fluid, breast milk, buccal mucosa, bile, chorionic villi, cytological specimens, embryonic cells, endocrine glands, epithelia, feces, fetal cells, gastric lavage fluid, heart, intestine, kidney, lavage fluid (e.g. bronchoalveolar fluid, ductal lavage fluid, ear lavage fluid, peritoneal fluid, arthroscopic fluid), liver, lymph node, lymphatic fluid, muscle, nasal mucus, peritoneal lavage fluid, pituitary, prostate fluid, semen, spleen, spinal fluid, sputum, sweat, swabs, tears, umbilical cord blood, vascular, whole blood.

[0158] In certain embodiments, the biological sample is derived from the group consisting of brain, spleen, bone, heart, vascular, lung, kidney, liver, intestine, muscle, blood, pituitary, endocrine glands, lymph node, epithelia, buccal mucosa, serum, plasma, cerebrospinal fluid, urine, saliva, biopsies, swabs and cytological specimens.

[0159] The term biological sample includes environmental samples and / or environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and nondisposable items.

[0160] A biological sample may be a purified RNA sample, a purified nucleic acid sample or an unpurified sample containing nucleic acids. Thus, in a particular embodiment, the invention relates to the method of the invention, wherein the biological sample is a purified nucleic acid sample, preferably wherein the purified nucleic acid sample comprises RNA or is suspected to comprise RNA.

[0161] Nucleic acids may be purified from different types of biological samples as known in the art, for example by using commercial kits.

[0162] In certain embodiments, the invention relates to a method according to the invention, wherein the biological sample is a sample that contains nucleic acids or is suspected to contain nucleic acids, preferably RNAs.

[0163] In certain embodiments, the invention relates to a method according to the invention, wherein the biological sample is a sample that contains nucleic acids, preferably a purified nucleic acid sample, more preferably wherein the nucleic acid has been purified from cells or viruses.

[0164] In a particular embodiment, the biological sample may be a sample that is suspected to contain viral RNA. In such embodiments, the method of the invention may be used to detect the presence of viruses in a sample and to quantify the viral load in a sample.

[0165] In certain embodiments, the invention comprises a method, wherein the biological sample is an unpurified sample containing nucleic acids, for example, a mixture of RNA and DNA.

[0166] Biological samples may include, but are not limited to, tissue samples from animals and plants, such as liver, brain, heart, muscle, leaves, roots, stems, and flowers. Additionally, cell cultures, including mammalian cell lines (e.g., HeLa, HEK293, CHO) and primary cells isolated directly from tissues (e.g., primary hepatocytes, neurons, immune cells), are suitable for analysis. Blood and blood-derived samples, such as whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, plasma, and serum, can also be analyzed to investigate systemic responses, immune function, and disease biomarkers. Clinical samples, including tissue biopsies (e.g., tumor biopsies, liver biopsies) and swabs or lavages (e.g., nasal, throat, vaginal, bronchoalveolar lavage), are applicable for detecting pathogens and studying local gene expression responses. Furthermore, microbial samples from bacterial and fungal cultures, as well as environmental samples from soil and water, are included to study microbial communities and environmental stress responses. Viral RNA from various viruses (e.g., SARS- CoV-2, influenza) is also encompassed for quantifying viral load and gene expression during infection.

[0167] Another aspect of the invention relates to the plurality of oligonucleotides that may be used in a reverse transcription reaction to produce tagged cDNAs. The same limitations and considerations described above for the method also apply, mutatis mutandis, to these embodiments directed to the plurality of oligonucleotides.

[0168] That is, in a particular embodiment, the invention relates to a plurality of oligonucleotides having the format 5'-X-(L)-B-3', wherein X is a unique sequence tag, L is an optional linker, and B is a random nucleotide sequence having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B).

[0169] In a particular embodiment, the invention relates to the plurality of oligonucleotides of the invention, wherein the random nucleotide sequence (B) has a length of 4 to 9 nucleotides, preferably 5 to 7 nucleotides, most preferably 6 nucleotides.

[0170] In a particular embodiment, the invention relates to the plurality of oligonucleotides of the invention, wherein the plurality of oligonucleotides comprises at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000 or at least 4000 different random nucleotide sequences (B).

[0171] In a particular embodiment, the invention relates to the plurality of oligonucleotides of the invention, wherein the linker L is a nucleic acid linker. In a particular embodiment, the invention relates to the plurality of oligonucleotides of the invention, wherein the unique sequence tag (X) is identical in all oligonucleotides comprised in the plurality of oligonucleotides.

[0172] In a particular embodiment, the invention relates to the plurality of oligonucleotides of the invention, wherein the sequence of the unique sequence tag (X) is absent in a target organism.

[0173] In a particular embodiment, the invention relates to the plurality of oligonucleotides of the invention, wherein the unique sequence tag (X) has a length of 6 to 80 nucleotides, preferably 10 to 50 nucleotides, more preferably 15 to 30 nucleotides.

[0174] In a particular embodiment, the invention relates to the plurality of oligonucleotides of the invention, wherein the unique sequence tag (X) comprises a sequence selected from the group consisting of: a) 5'-TAG CCG TAA TCG TAG GAT CG-3' (SEQ ID NO: 1); b) 5'-CCG ATC ACC TTT AGA GCC AA-3' (SEQ ID NO: 2); c) 5'-CGC ACA TTA TTT TAG TGG GGT CAT GAG GTT AGG GAT GTT T-3' (SEQ ID NO: 3); d) 5'-GGG TGA TGC CCG TGC AAA TCA AAC TCT CGG AGA AGG CGA TAG GTT TTA CGG ATT TTT AGT-3' (SEQ ID NO: 4).

[0175] A further aspect of the present invention relates to a kit comprising the plurality of oligonucleotides of the invention.

[0176] In certain embodiments, the kit may further comprise one or more of: a reverse transcriptase enzyme, an RNAse H enzyme, a DNA polymerase, a buffer, at least one target-specific forward primer, at least one reverse primer that is at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X), a nucleic acid dye, and / or a nucleic acid probe comprising a detectable marker.

[0177] It is to be understood that kits comprising one or more target-specific forward primer(s) and / or nucleic acid probe(s) may be limited to the detection and quantification of specific target nucleic sequence. Alternatively, target-specific forward primers and / or nucleic acid probes may be designed individually based on the target nucleic acid that is to be detected and quantified.

[0178] All numeric ranges provided herein are inclusive of narrower ranges; delineated upper and lower range limits are interchangeable to create further ranges not explicitly delineated. The number of significant digits conveys neither limitation on the indicated amounts nor on the accuracy of the measurements.

[0179] In this document, the terms "a" or "an" are used to include one or more than one and the term "or" is used to refer to a nonexclusive "or" unless otherwise indicated.

[0180] The term "about," as used herein, means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0181] BRIEF DESCRIPTION OF DRAWINGS

[0182] Figure 1 shows comparison of Ct values in RT-qPCR for GAPDH in HepG2 cells, carried out either with a commercial kit obtained from Thermo Fisher Scientific using as primer the random hexamers described in Froussard P. (1993) ("Commercial Kit") or with the kit of the present invention ("Present Kit") comprising the plurality of oligonucleotides described herein. A) shows the amplification curve for the GAPDH transcript along with the respective negative-RT reaction control. B) A significant Ct value was obtained in the negative-RT control of the Commercial kit but not in negative-RT control of the kit of the present invention.

[0183] Figure 2 shows that DNA contamination does not affect the kit of the present invention ("Present Kit")-based quantification of gene expression on RNA extracted form HeLa cells. A) DNase-treated negative-RT control samples do not give any signal, whether the kit obtained from Thermo Fisher Scientific using as primer the random hexamers described in Froussard P. (1993) ("Commercial Kit") or the kit of the present invention ("Present Kit") are used. A detectable Ct value is obtained when the "Commercial Kit" is used on a sample not treated with DNase. The "Present Kit" gives similar signal irrespective of the DNase treatment. B) Ct values inferred from each of the amplification curves shown in A.

[0184] Figure 3 shows that the kit of the present invention ("Present Kit") comprising the plurality of oligonucleotides described herein allows for screening of multiple transcripts from the same cDNA reaction. RT-qPCR for mCherry in HepG2 cell, comparing the Ct values obtained with the kit obtained from Thermo Fisher Scientific using as primer the random hexamers described in Froussard P. (1993) ("Commercial Kit") and the kit of the present invention ("Present Kit"). A) Amplification curve for the mCherry transcript along with the respective negative-RT reaction control. B) A significant Ct value was determined in the negative-RT control of the commercial kit (B) but not in the negative-RT control of the kit of the present invention ("Present Kit").

[0185] EXAMPLES

[0186] Materials and methods cDNA sample preparation:

[0187] RNA was extracted from HeLa (ATCC# CCL-2™) or HepG2 cells (ATCC# HB-8065) (expressing mCherry via high copy number plasmid (Takara# 631630) grown at 80% confluency in individual wells of a standard 6-well plate, with Trizol RNA extraction reagent (Thermo Fisher) according to the manufacturer's protocol. One part of the extracted RNA was treated with a higher amount of DNAse (TurboDNAase, Thermo) than recommended in manufacturer's protocol, to ensure the removal of contaminating DNA. lOug of each DNase-untreated and DNase-treated RNA were used to prepare cDNA. This was done using as primer either random hexamers as described in Froussard P, 1993 or a plurality of oligonucleotides according to the invention consisting of the same random hexamers as random nucleotide sequence (B) which binds to nucleotides in the RNA of an organism (human RNA in this experiment) tagged with TAG CCG TAA TCG TAG GAT CG (SEQ ID NO: 1) as a unique sequence tag (X) (human RNA in this experiment) on the 5' end with no linker nucleotide sequence between the random nucleotide sequence (B) and the a unique sequence tag (X). The samples were then treated with RNase H according to the protocol of Superscript IV reverse transcriptase (Thermo Fisher) to remove the hybridized RNA, and the treated cDNA was purified using Nucleospin PGR cleanup kit (Macherey Nagel). The cDNA concentration was measured using nanodrop (Thermo Fisher). qPCR:

[0188] Power SYBR green kit (Thermo Fisher) was used for the qPCR reaction in AB QuantStudio 3. The reaction mix was as follows.

[0189] Reaction volume: 20ul

[0190] Power SYBRGreen (Thermo) 2X: lOul

[0191] Forward primer for GAPDH: AGA AGG CTG GGG CTC ATT TG (SEQ ID NO: 5) / forward primer for mCherry: CAT CCC CGA CTA CTT GAA GC (SEQ ID NO: 6) lOuM: 0.4ul Reverse primer for GAPDH: TTA CTC CTT GGA GGC CAT GT (SEQ ID NO: 7) / mCherry:

[0192] CCC ATG GTC TTC TTC TGC AT (SEQ ID NO: 8) / Unique primer: TAG CCG TAA TCG

[0193] TAC GAT CG (SEQ ID NO: 1) lOuM: 0.4ul cDNA: 2ul (20 ng)

[0194] Nuclease free water: 7.2ul

[0195] The reaction was cycled using the standard cycling condition of qPCR on AB Quantstudio 3 (melting at 95 degrees Celsius for 10 seconds and extension 60 degrees Celsius for 1 minute) for 40 cycles.

[0196] Example 1

[0197] To test the efficiency of the method of the invention, the inventors extracted RNA from mCherry-expressing HepG2 cells and converted it into cDNA using either random hexamer primers as described in Froussard P, 1993 or the above-described plurality of oligonucleotides according to the invention consisting of a random nucleotide sequence (B) and a unique sequence tag (X) on the 5' end comprising the sequence TAG CCG TAA TCG TAC GAT CG (SEQ ID NO: 1), without DNase treatment. The cDNA was then purified and used to measure the expression of the GAPDH gene by qPCR. In the qPCR method using as primer the random hexamers described in Froussard P, 1993, the inventors used forward and reverse primers specific to the gene of interest as described above to amplify the signal of the gene, while in the method of the present invention the inventors used the same forward primer but as the reverse primer RT primer according to the invention as described above. The two methods showed GAPDH amplification to different Ct values (Fig 1A, B), lower for the method using as primer the random hexamers described in Froussard P, 1993, which may be explained by the DNA contamination carried to the cDNA samples.

[0198] Example 2

[0199] To confirm that DNA contamination contributes to the lower Ct value (and thus higher estimated abundance of the target GAPDH RNA) in the method using as primer the random hexamers described in Froussard P, 1993 the inventors also setup the negative RT reaction, in which RNA is used in the qPCR reaction without prior conversion to cDNA. As only DNA can be amplified during qPCR, the amplification from negative RT reaction only comes from the DNA contamination. Thus, this reaction reveals whether DNA indeed contaminated the initial sample.

[0200] The amplification plots and Ct values from negative-RT reactions showed that the method using as primer the random hexamers described in Froussard P, 1993 also amplifies signals from DNA contamination (Fig. 1A, B), while the method of the present invention only amplifies the RNA-derived cDNA signals, being completely insensitive to DNA contamination in the initial sample. Further, to validate that presence of DNA contamination does not affect the quantification of the gene by the method of the present invention, the inventors treated RNA samples with DNase to the extent where no signal was amplified from the genomic DNA in the negative-RT reaction with method using as primer the random hexamers described in Froussard P, 1993 (Fig. 2A and B). Similar Ct values were obtained with the method of the invention whether or not the sample was DNase-treated. In contrast, the DNase treatment led to a slight but significant increase in the Ct value obtained with the method using as primer the random hexamers described in Froussard P, 1993 again emphazing that DNA contamination affects the quantification of RNAs by RT-qPCR.

[0201] Example 3

[0202] GAPDH is a highly expressed endogenous gene. The issue of DNA contamination should be exacerbated by decreased RNA to DNA abundance. This occurs when the gene of interest is expressed from a plasmid, as plasmids are generally present in many copies per cell. To test whether the method of the invention can withstand the higher contamination as well, the inventors isolated RNA from HepG2 cells that expressed the mCherry gene from a high copynumber plasmid. The Inventors measured the mCherry expression using again the method using as primer the random hexamers described in Froussard P, 1993 and the methods of the invention and found that the method of the invention did not amplify signals from plasmid DNA, as can be seen from the negative-RT reaction (Fig 3 And B). In addition, the mCherry transcript was quantified using the same cDNA mix as of GAPDH (Fig 1 A and B), showing that using the plurality of oligonucleotides of the invention, one can quantify multiple transcripts from the same cDNA mix. Discussion

[0203] Without being bound by theory, the basic principle behind the method of the invention is to tag the cDNA, and thus distinguish it from the genomic or plasmid DNA. This is achieved by tagging the plurality of oligonucleotides with a unique sequence that is not present in the genomic DNA and is necessary for the amplification of any RNA signal. This leads to dependable omission of the contaminating DNA amplification. To test the principle, the inventors have isolated RNA from HepG2 cells transfected with mCherry-encoding plasmid using the Trizol method. cDNA was produced by the regular RT reaction using random hexamers as described in Froussard P, 1993 and primer mix comprising the plurality of oligonucleotides of the invention. The qPCR results shown in Fig 1 indicate that the normal RT-qPCR does not discriminate between genomic / plasmid DNA and RNA, while the method of the invention clearly does. To validate that the difference in Ct-value obtained with the regular RT-qPCR method and the method of the invention is indeed coming from the presence of contaminating DNA in the method using as primer the random hexamers described in Froussard P, 1993, the inventors treated the samples with TurboDNAse. This led to a drop in the RT-qPCR signal in the method using as primer the random hexamers described in Froussard P, 1993, but no change in the method of the invention. Moreover, the inventors also found that the method of the invention can be used to quantify multiple targets from the same initial sample. A technology of this nature is of immense research and diagnostic value. In a research lab, this technology allows the scientist to obtain accurate estimates of RNA abundance in a fraction of the time they need today.

[0204] References

[0205] Froussard P. rPCR: a powerful tool for random amplification of whole RNA sequences. PCR Methods Appl. 2(3):185-190 (1993). DOI: 10.1101 / gr.2.3.185.

[0206] Heid CA, Stevens J, Livak KL, Williams PM. Real time quantitative PCR. Genome Res.

[0207] 6(10):986-994 (1996). DOI: 10.1101 / gr.6.10.986.

[0208] Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1:263-273 (1986). DOI: 10.1101 / sqb.l986.051.01.032.

[0209] Sambrook J. Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells.

[0210] In Sambrook, J., Fritsch, E. R., & Maniatis, T. (eds). Molecular Cloning: A Laboratory

[0211] Manual (2nd ed). Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, vol. 1, ch. 7 (1989).

[0212] Smith CJ, Osborn AM. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol. 67(l):6-20 (2009). DOI: 10.1111 / j.1574-6941.2008.00629.x.

[0213] Tavares L, Alves PM, Ferreira RB, Santos CN. Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma. BMC Res Notes. 4:3 (2011). DOI: 10.1186 / 1756-0500-4-3.

[0214] Wan H, Seth A, Rainen L, Fernandes H. Coamplification of HIV-l Proviral DNA and Viral

[0215] RNA in Assays Used for Quantification of HIV-1 RNA. J Clin Microbiol. 48(6): 2186-2190

Claims

PCT-Patent Application based on 24 199 354.2Universitat BaselVossius Ref.: AJ2918 PCT BSCLAIMS1. A method for detecting and quantifying at least one target nucleic acid in a reverse transcriptase polymerase chain reaction (RT-PCR), the method comprising: a) reverse transcribing RNA present in a biological sample into complementary DNA (cDNA) using a plurality of oligonucleotides, wherein the oligonucleotides in the plurality of oligonucleotides comprise(i) a random nucleotide sequence (B) at the 3' end having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B); and(ii) a unique sequence tag (X) at the 5' end; b) amplifying the tagged cDNA obtained in step (a) using a pair of primers, wherein the forward primer specifically anneals to a target sequence within the cDNA and wherein the reverse primer comprises a nucleic acid sequence that is at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X); and c) detecting and quantifying the nucleic acids amplified in step (b).

2. The method of claim 1, wherein step (a) results in reverse transcription of the entire RNA in the biological sample into cDNA.

3. The method of claim 1 or 2, wherein the method does not require a step of purifying the cDNA obtained in step (a) before the amplification in step (b).

4. The method of any one of claims 1 to 3, wherein the detection and quantification of the nucleic acids amplified in step (b) comprises the use of a nucleic acid dye; or wherein the detection and quantification of the nucleic acids amplified in step (b) comprises the use of a nucleic acid probe comprising a detectable marker, in particular wherein the nucleic acid probe is designed such that the detectable marker is released from the nucleic acid probe when the cDNA is amplified from the forward or reverse primer and / or wherein the detectable marker is a fluorescent marker, preferably wherein the nucleic acid probe further comprises a quencher that decreases the fluorescence intensity of the fluorescent marker when bound to the nucleic acid probe.

5. The method of any one of claims 1 to 4, wherein two or more pairs of primers are added in step (b), preferably wherein the forward primers of the two or more primers specifically anneal to different target sequences within the cDNA and wherein the reverse primers of the two or more primers comprise a nucleic acid sequence that is at least partially identical to the nucleic acid sequence of the unique sequence tag, in particular wherein the tagged cDNA obtained in step (a) is divided into two or more samples before the amplification step (b) and a pair of primers is added to each sample.

6. A method for generating a plurality of tagged cDNA molecules, the method comprising: a) providing a biological sample comprising RNA; b) adding a plurality of oligonucleotides to the biological sample, wherein the oligonucleotides in the plurality of oligonucleotides comprise(i) a random nucleotide sequence (B) at the 3' end having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B); and(ii) a unique sequence tag (X) at the 5' end; and c) reverse transcribing the RNA in the biological sample into cDNA with a reverse transcriptase to obtain a plurality of tagged cDNA molecules.

7. The method of any one of claims 1 to 6, wherein the oligonucleotides in the plurality of oligonucleotides have the format 5'-X-(L)-B-3', wherein X is the unique sequence tag, L is an optional linker, and B is the random nucleotide sequence, in particular wherein the linker L is a nucleic acid linker.

8. The method of any one of claims 1 to 7, wherein the random nucleotide sequence (B) has a length of 4 to 9 nucleotides, preferably 5 to 7 nucleotides, most preferably 6 nucleotides; and / or wherein the plurality of oligonucleotides comprises at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000 or at least 4000 different random nucleotide sequences (B).

9. The method of to any one of claims 1 to 8, wherein the plurality of oligonucleotides is a pool of random oligomers, wherein each random oligomer is linked to a unique sequence tag (X) at its 5' end, and / or wherein the unique sequence tag (X) is identical in all oligonucleotides comprised in the plurality of oligonucleotides.

10. The method of any one of claims 1 to 9, wherein the sequence of the unique sequence tag (X) is absent in the organism from which the biological sample is derived.

11. The method of any one of claims 1 to 10, wherein the unique sequence tag (X) has a length of 6 to 80 nucleotides, preferably 10 to 50 nucleotides, more preferably 15 to 30 nucleotides, and / or wherein the unique sequence tag (X) comprises a sequence selected from the group consisting of: a) 5'-TAG CCG TAA TCG TAG GAT CG-3' (SEQ ID NO: 1); b) 5'-CCG ATC ACC TTT AGA GCC AA-3' (SEQ ID NO: 2); c) 5'-CGC ACA TTA TTT TAG TGG GGT CAT GAG GTT AGG GAT GTT T-3' (SEQ ID NO: 3); d) 5'-GGG TGA TGC CCG TGC AAA TCA AAC TCT CGG AGA AGG CGA TAG GTT TTA CGG ATT TTT AGT-3' (SEQ ID NO: 4).

12. The method of any one of claims 1 to 11, wherein the biological sample is a purified nucleic acid sample, preferably wherein the purified nucleic acid sample comprises RNA or is suspected to comprise RNA, in particular wherein the purified RNA sample is derived from the group consisting of: brain, spleen, bone, heart, vascular, lung, kidney, liver, intestine, muscle, blood, pituitary, endocrine glands, lymph node, epithelia, buccal mucosa, serum, plasma, cerebrospinal fluid, urine, saliva, biopsies, swabs and cytological specimens.

13. A plurality of oligonucleotides having the format 5'-X-(L)-B-3', wherein X is a unique sequence tag, L is an optional linker, and B is a random nucleotide sequence having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B).

14. The plurality of oligonucleotides of claim 13, wherein the random nucleotide sequence (B) has a length of 4 to 9 nucleotides, preferably 5 to 7 nucleotides, most preferably 6 nucleotides; and / or wherein the plurality of oligonucleotides comprises at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000 or at least 4000 different random nucleotide sequences (B); and / or wherein the linker L is a nucleic acid linker; and / or wherein the unique sequence tag (X) is identical in all oligonucleotides comprised in the plurality of oligonucleotides; and / or wherein the sequence of the unique sequence tag (X) is absent in a target organism; and / or wherein the unique sequence tag (X) has a length of 6 to 80 nucleotides, preferably 10 to 50 nucleotides, more preferably 15 to 30 nucleotides; and / orwherein the unique sequence tag (X) comprises a sequence selected from the group consisting of: a) 5'-TAG CCG TAA TCG TAG GAT CG-3' (SEQ ID NO: 1); b) 5'-CCG ATC ACC TTT AGA GCC AA-3' (SEQ ID NO: 2); c) 5'-CGC ACA TTA TTT TAG TGG GGT CAT GAG GTT AGG GAT GTT T-3' (SEQ ID NO: 3); d) 5'-GGG TGA TGC CCG TGC AAA TCA AAC TCT CGG AGA AGG CGA TAG GTT TTA CGG ATT TTT AGT-3' (SEQ ID NO: 4).

15. A kit comprising the plurality of oligonucleotides of any one of claims 13 or 14, in particular wherein the kit further comprises one or more of: a reverse transcriptase enzyme, an RNAse H enzyme, a DNA polymerase, a buffer, at least one target-specific forward primer, at least one reverse primerthat is at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X), a nucleic acid dye, and / or a nucleic acid probe comprising a detectable marker.

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