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KR-102960736-B1 - Depletion of unwanted RNA species

KR102960736B1KR 102960736 B1KR102960736 B1KR 102960736B1KR-102960736-B1

Abstract

The present disclosure provides a method and kit for inhibiting the synthesis of cDNA of unwanted RNA species during reverse transcription. The method and kit provided herein use a blocking oligonucleotide, such as a lock nucleic acid (LNA).

Inventors

  • 샤퍼, 조나단
  • 라더, 에릭
  • 톨스트루프, 니엘스
  • 크룸헤우에르, 외르크
  • 킴, 다니엘 와이.
  • 스트라우스, 자샤

Assignees

  • 퀴아젠 사이언시스, 엘엘씨

Dates

Publication Date
20260507
Application Date
20190919
Priority Date
20180925

Claims (20)

  1. A method for inhibiting cDNA synthesis of one or more unwanted RNA species in an RNA sample during reverse transcription, comprising the following steps: (a) a step of providing an RNA sample comprising one or more desired RNA species and one or more unwanted RNA species, (b) a step of annealing one or more blocking oligonucleotides to one or more regions of one or more unwanted RNA species within an RNA sample to produce a template mixture, and Here, one or more blocking oligonucleotides bind stably and complementarily to one or more regions of one or more unwanted RNA species, and include a 3' modification that prevents the one or more blocking oligonucleotides from being extended, and Here, the number of one or more blocking oligonucleotides is at least 10, and Here, two or more of the blocking oligonucleotides anneale into different regions of at least one of one or more unwanted RNA species, and Herein, a step in which the distance between two adjacent regions of at least one of one or more unwanted RNA species being annealed by two or more blocking oligonucleotides is in the range of 0 to 100 nucleotides, 0 to 75 nucleotides, 0 to 50 nucleotides, 20 to 100 nucleotides, 20 to 75 nucleotides, 20 to 50 nucleotides, 30 to 100 nucleotides, 30 to 75 nucleotides, 30 to 50 nucleotides, or 30 to 45 nucleotides; and (c) a step comprising incubating a template mixture with a reaction mixture comprising, under conditions sufficient to synthesize a cDNA molecule using one or more desired RNA species as template(s), wherein cDNA synthesis using one or more unwanted RNA species is inhibited: (i) at least one reverse transcriptase, (ii) one or more reverse transcription primers, and (iii) Reaction buffer.
  2. A method according to claim 1, wherein at least one of the one or more blocking oligonucleotides or each comprises one or more modified nucleotides that increase binding between one or more blocking oligonucleotides and one or more regions of unwanted RNA species.
  3. A method of claim 1, wherein at least one of the one or more blocking oligonucleotides or each of them does not include any modified nucleotide that increases binding between one or more blocking oligonucleotides and one or more regions of unwanted RNA species, and is at least 25 nucleotides long.
  4. A method according to claim 2, wherein at least one or each of the one or more blocking oligonucleotides comprises one or more lock nucleic acids (LNAs), the number of LNAs within the one or more blocking oligonucleotides may be in the range of 2 to 20, 4 to 16, or 3 to 15, and the length of the one or more blocking oligonucleotides may be in the range of 10 to 30 nucleotides, 16 to 24 nucleotides, or 18 to 22 nucleotides.
  5. A method that satisfies one or more of the following (1) to (14) in any one of paragraphs 1 to 4: (1) The melting temperature (Tm) of the duplex formed between one or more blocking oligonucleotides and one or more regions of one or more unwanted RNA species is in the range of 80 to 96°C or 86 to 92°C; (2) The number of one or more blocking oligonucleotides is at least 50, at least 100, at least 150, 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 1500, 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 10,000, maximum 100,000, maximum 90,000, maximum 80,000, maximum 70,000, maximum 60,000, maximum 50,000, 10 to 100,000, 100 to 80,000, or 800 to It is 50,000; (3) The number of one or more unwanted RNA species annealed by one or more blocking oligonucleotides is at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, max. 1,000,000, max. 500,000, max. 100,000, max. 50,000, max. 10,000, max. 9,000, max. 8,000, max. 7,000, max. 6,000, max. 5,000, max. 4,000, max. 3,000, max. 2,000, 2 to 1,000,000, 100 to 500,000, 500 to 100,000, or is 1,000 to 10,000; (4) One or more unwanted RNA species contain rRNA; (5) One or more unwanted RNA species include 28S rRNA, 18S rRNA, 5.8S rRNA, eukaryotic 5S rRNA, mitochondrial 12S rRNA, mitochondrial 16S rRNA, plastid rRNA, prokaryotic 5S rRNA, prokaryotic 16S rRNA or prokaryotic 23S rRNA; (6) One or more unwanted RNA species include an abundant protein-coding mRNA, tRNA, snoRNA, or snRNA, and the abundant protein-coding mRNA may be globin RNA; (7) Step (b) is performed in the presence of a salt, wherein the salt may be KCl, or the concentration of the salt in the template mixture of step (b) may be in the range of 5 mM to 50 mM, 10 mM to 30 mM, or 15 mM to 25 mM; (8) The amount of each of one or more blocking oligonucleotides in the template mixture of step (b) is in the range of 0.1 pmol to 50 pmol, 0.5 pmol to 20 pmol, 0.5 pmol to 10 pmol, 1 pmol to 20 pmol, 1 pmol to 10 pmol, 1.5 pmol to 10 pmol, 1.5 pmol to 8 pmol, or 2 pmol to 7 pmol per blocking oligonucleotide; (9) Step (b) (i) a step of contacting one or more blocking oligonucleotides with an RNA sample, (ii) a step of incubating the mixture of step (i) at least 65°C, at least 70°C, or at least 75°C for at least 30 seconds, at least 1 minute, or at least 2 minutes, and (iii) After step (ii), a step of reducing the temperature to less than 40℃ or less than 25℃ Includes; (10) One or more reverse transcription primers are random primers or random hexamers; (11) RNA samples contain fragmented RNA molecules; (12) RNA samples are prepared from whole blood, serum, or plasma; (13) The method (d) A step of synthesizing a complementary strand of the cDNA molecule produced in step (c) to produce a double-stranded cDNA molecule; (e) a step of amplifying double-stranded cDNA molecules to construct a sequence analysis library; and (f) A step of sequencing one or more desired RNA species using the sequencing library constructed in step (e). additionally including one of; and (14) One or more blocking oligonucleotides are completely complementary to one or more regions of one or more unwanted RNA species.
  6. A method in paragraph 1 that satisfies either (i) or (ii) below: (i) at least one different region of one or more unwanted RNA species is evenly distributed, and the distance between two adjacent regions is in the range of 20 to 50 nucleotides or 30 to 45 nucleotides; or (ii) At least one different region of one or more unwanted RNA species is not evenly distributed.
  7. A set of blocking oligonucleotides that are complementary or completely complementary to multiple regions of an unwanted RNA species, wherein each blocking oligonucleotide comprises one or more modified nucleotides that increase its binding to a region of the unwanted RNA species, and the number of blocking oligonucleotides in the set is at least 10, wherein at least two of the blocking oligonucleotides anneale different regions of the unwanted RNA species, and wherein the distance between two adjacent regions of the unwanted RNA species annealed by the set of blocking oligonucleotides is in the range of 0 to 100 nucleotides, 0 to 75 nucleotides, 0 to 50 nucleotides, 20 to 100 nucleotides, 20 to 75 nucleotides, 20 to 50 nucleotides, 30 to 100 nucleotides, 30 to 75 nucleotides, 30 to 50 nucleotides, or 30 to 45 nucleotides.
  8. In claim 7, a set of blocking oligonucleotides satisfying one or more of the following (1) to (8): (1) Each blocking oligonucleotide contains a 3' modification that prevents it from being extended; (2) Each blocking oligonucleotide comprises one or more lock nucleic acids (LNAs), wherein the number of LNAs within the blocking oligonucleotide may be in the range of 2 to 20, 4 to 16, or 3 to 15; (3) The length of the blocking oligonucleotide is in the range of 10 to 30 nucleotides, 16 to 24 nucleotides, or 18 to 22 nucleotides; (4) The melting temperature (Tm) of the duplex formed between the blocking oligonucleotide and the region of the unwanted RNA species is in the range of 80 to 96°C or 86 to 92°C; (5) The number of blocking oligonucleotides in the set is at least 20, at least 30, at least 40, at least 50, at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, 10 to 1000, 10 to 500, or 10 to 300; (6) Blocking oligonucleotides are complementary to evenly spaced regions of unwanted RNA species; (7) Unwanted RNA species include rRNA, 28S rRNA, 18S rRNA, 5.8S rRNA, eukaryotic 5S rRNA, mitochondrial 12S rRNA, mitochondrial 16S rRNA, plastid rRNA, prokaryotic 5S rRNA, prokaryotic 16S rRNA or prokaryotic 23S rRNA; and (8) Unwanted RNA species are abundant protein-coding mRNA, globin RNA, tRNA, snoRNA, or snRNA.
  9. A plurality of sets of blocking oligonucleotides, each set being in accordance with paragraph 7.
  10. A plurality of sets of blocking oligonucleotides, each set being in accordance with paragraph 8.
  11. In claim 9, a plurality of sets of blocking oligonucleotides satisfying one or more of the following (1) to (7): (1) Different sets of blocking oligonucleotides anneale regions of different unwanted RNA species; (2) The number of different unwanted RNA species is at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at most 1,000,000, at most 500,000, at most 100,000, at most 50,000, at most 10,000, at most 9,000, at most 8,000, at most 7,000, at most 6,000, at most 5,000, at most 4,000, at most 3,000, at most 2,000, 2 to 1,000,000, 100 to 500,000, 500 to 100,000, or 1,000 to It is 10,000; (3) The number of sets of blocking oligonucleotides is at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at most 10,000, at most 9,000, at most 8,000, at most 7,000, at most 6,000, at most 5,000, at most 4,000, at most 3,000, at most 2,000, 2 to 10,000, 2 to 5,000, 2 to 1000, 2 to 500, 2 to 200, 10 to 10,000, 10 to 5,000, 10 to 1000, 10 to 500, 10 to 200, 100 to 10,000, 100 to 5,000, 100 to 1,000, or 100 to 500; (4) The total number of blocking oligonucleotides in a plurality of sets is at least 50, at least 100, at least 150, 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 1500, 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 10,000, maximum 100,000, maximum 90,000, maximum 80,000, maximum 70,000, maximum 60,000, maximum 50,000, 20 to 100,000, 100 to 80,000, or 800 to It is 50,000; (5) Different unwanted RNA species are from a single organism; (6) Unwanted RNA species include rRNA, 28S rRNA, 18S rRNA, 5.8S rRNA, eukaryotic 5S rRNA, mitochondrial 12S rRNA, mitochondrial 16S rRNA, plastid rRNA, prokaryotic 5S rRNA, prokaryotic 16S rRNA or prokaryotic 23S rRNA; and (7) Unwanted RNA species include protein-coding mRNA, globin RNA, tRNA, snoRNA, or snRNA.
  12. In item 10, a plurality of sets of blocking oligonucleotides satisfying one or more of the following (1) to (7): (1) Different sets of blocking oligonucleotides anneale regions of different unwanted RNA species; (2) The number of different unwanted RNA species is at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at most 1,000,000, at most 500,000, at most 100,000, at most 50,000, at most 10,000, at most 9,000, at most 8,000, at most 7,000, at most 6,000, at most 5,000, at most 4,000, at most 3,000, at most 2,000, 2 to 1,000,000, 100 to 500,000, 500 to 100,000, or 1,000 to It is 10,000; (3) The number of sets of blocking oligonucleotides is at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at most 10,000, at most 9,000, at most 8,000, at most 7,000, at most 6,000, at most 5,000, at most 4,000, at most 3,000, at most 2,000, 2 to 10,000, 2 to 5,000, 2 to 1000, 2 to 500, 2 to 200, 10 to 10,000, 10 to 5,000, 10 to 1000, 10 to 500, 10 to 200, 100 to 10,000, 100 to 5,000, 100 to 1,000, or 100 to 500; (4) The total number of blocking oligonucleotides in a plurality of sets is at least 50, at least 100, at least 150, 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 1500, 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 10,000, maximum 100,000, maximum 90,000, maximum 80,000, maximum 70,000, maximum 60,000, maximum 50,000, 20 to 100,000, 100 to 80,000, or 800 to It is 50,000; (5) Different unwanted RNA species are from a single organism; (6) Unwanted RNA species include rRNA, 28S rRNA, 18S rRNA, 5.8S rRNA, eukaryotic 5S rRNA, mitochondrial 12S rRNA, mitochondrial 16S rRNA, plastid rRNA, prokaryotic 5S rRNA, prokaryotic 16S rRNA or prokaryotic 23S rRNA; and (7) Unwanted RNA species include protein-coding mRNA, globin RNA, tRNA, snoRNA, or snRNA.
  13. In paragraph 11, a plurality of sets of blocking oligonucleotides satisfying one or more of the following (i) to (ii): (i) different unwanted RNA species are from a plurality of different organisms, and the number of different organisms may be at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at most 10,000, at most 5,000, at most 1,000, at most 500, at most 100, 2 to 10,000, 5 to 5,000, or 10 to 1,000; and (ii) Different unwanted RNA species are the same type of unwanted RNA species.
  14. In Clause 12, a plurality of sets of blocking oligonucleotides satisfying one or more of the following (i) to (ii): (i) different unwanted RNA species are from a plurality of different organisms, and the number of different organisms may be at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at most 10,000, at most 5,000, at most 1,000, at most 500, at most 100, 2 to 10,000, 5 to 5,000, or 10 to 1,000; and (ii) Different unwanted RNA species are the same type of unwanted RNA species.
  15. A kit for inhibiting cDNA synthesis of one or more unwanted RNA species in an RNA sample, comprising the following: (1) (a) one or more blocking oligonucleotides that are complementary to one or more regions of one or more unwanted RNA species in an RNA sample, each comprising one or more modified nucleotides that increase binding between one or more blocking oligonucleotides and one or more regions of one or more unwanted RNA species, wherein the number of one or more blocking oligonucleotides is at least 10, wherein two or more of the blocking oligonucleotides anneale at least one different region of one or more unwanted RNA species, wherein the distance between two adjacent regions of at least one of one or more unwanted RNA species annealed by two or more blocking oligonucleotides is 0 to 100 nucleotides, 0 to 75 nucleotides, 0 to 50 nucleotides, 20 to 100 nucleotides, 20 to 75 nucleotides, 20 to 50 nucleotides, 30 to 100 nucleotides, 30 to 75 nucleotides, 30 to 50 nucleotides, or 30 One or more blocking oligonucleotides ranging from to 45 nucleotides, or (b) a set or multiple sets of blocking oligonucleotides of any one of paragraphs 7 to 14, and (2) Reverse transcriptase.
  16. A kit according to claim 15, further comprising one or more of the group consisting of a reverse transcription primer, a reverse transcription buffer, an enzyme for synthesizing a second cDNA strand, a DNA polymerase for PCR amplification, a ligase, a DNA polymerase for sequencing, an oligonucleotide primer for DNA amplification or sequencing, and an adapter.
  17. A method according to claim 1, wherein one or more blocking oligonucleotides of step (b) are designed by a method comprising the following steps: (A) A step of generating multiple blocking oligonucleotides complementary to regions of one or more unwanted RNA species, (B) A step of filtering out unallowed blocking oligonucleotides, (C) a step of generating one or more groups of blocking oligonucleotides complementary to multiple different regions of one or more unwanted RNA species, and (D) A step of optionally shuffling blocking oligonucleotides among the groups to create a new group of blocking oligonucleotides, and selecting one or more of the new groups of blocking oligonucleotides.
  18. In paragraph 17, a method satisfying one or more of the following (1) to (8): (1) Multiple distinct regions of one or more unwanted RNA species are evenly spaced along one or more unwanted RNA species; (2) Each of the multiple blocking oligonucleotides includes a 3' modification that prevents the multiple blocking oligonucleotide from being extended; (3) Each of the multiple blocking oligonucleotides comprises one or more modified nucleotides that increase binding between the blocking oligonucleotide and one or more regions of unwanted RNA species, and each of the multiple blocking oligonucleotides may comprise one or more lock nucleic acids (LNA); (4) The distance between adjacent regions of one or more unwanted RNA species is in the range of 20 to 50, 25 to 50, 30 to 50, 20 to 45, 25 to 45, 30 to 45, or 31 to 43 nucleotides; (5) The method comprises step (D), wherein the number of blocking oligonucleotides in at least one of the new groups selected in step (D) is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, 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 most 10,000, at most 9,000, at most 8,000, at most 7,000, at most 6,000, at most 5,000, 10 to 10,000, or 100 to 5,000 different blocking oligonucleotides; (6) The number of one or more unwanted RNA species is at least 100, and step (C) uses a greedy algorithm to generate one or more groups of blocking oligonucleotides; (7) further comprises experimentally testing the efficacy of the method in inhibiting the synthesis of cDNA of one or more unwanted RNA species or the off-target depletion of one or more groups of blocking oligonucleotides generated in step (C) or selected in step (D); and (8) The multiple blocking oligonucleotides of step (A) are completely complementary to the region of one or more unwanted RNA species.
  19. In claim 18, a method wherein a multi-blocking oligonucleotide satisfies (3) above and has one or more of the following features: (i) The length of the multiple blocking oligonucleotide is in the range of 10 to 30 nucleotides, 16 to 24 nucleotides, 17 to 23 nucleotides, or 18 to 22 nucleotides; (ii) The number of LNAs in each of the multiple blocking oligonucleotides is in the range of 2 to 20, 4 to 16, or 3 to 15; (iii) The melting temperature of the duplex formed between the multiple blocking oligonucleotide and the region of the multiple blocking oligonucleotide and the complementary unwanted RNA species is in the range of 80 to 96°C, 82 to 94°C, or 86 to 92°C; (iv) The number of multiple blocking oligonucleotides generated in step (A) is at least 100, 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, max. 1,000,000, max. 500,000, max. 100,000, max. 90,000, max. 80,000, max. 70,000, max. 60,000, max. 50,000, 100 to 1,000,000, 500 to 100,000, or 1,000 to 10,000; (v) Multiple blocking oligonucleotides may bind to regions of unwanted RNA species that are complementary to themselves rather than to themselves; (vi) The multiple blocking oligonucleotide is likely to bind to a region of the unwanted RNA species that is complementary to the unwanted RNA species(s) rather than to another region within the transcript to which the unwanted RNA species(s) belong; and (vii) The number of different unwanted RNA species that are complementary or completely complementary to the multiple blocking oligonucleotides is at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, max. 1,000,000, max. 500,000, max. 100,000, max. 50,000, max. 10,000, max. 9,000, max. 8,000, max. 7,000, max. 6,000, max. 5,000, max. 4,000, max. 3,000, max. 2,000, 2 to 1,000,000, 100 to 500,000, 500 to It is 100,000, or 1,000 to 10,000.
  20. delete

Description

Depletion of unwanted RNA species Statement regarding the sequence list The sequence list associated with this application is provided in text format instead of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence list is 830109_416WO_SEQUENCE_LISTING.txt. The text file is 97.5 KB, was created on September 15, 2019, and is submitted electronically via EFS-Web. Technology field The present disclosure relates particularly to a method and kit for depleting unwanted RNA species from an RNA sample for constructing a transcriptome sequencing library. Libraries constructed for transcriptome sequencing consist largely of unwanted species (e.g., cytoplasmic ribosomal RNA, mitochondrial ribosomal RNA, and globin mRNA) that account for the majority of the sequencing budget and make RNA sequencing extremely inefficient. rRNA alone constitutes more than 80% of the RNA found in samples. Consequently, various methods have been developed to enrich mRNA or deplete unwanted RNA from next-generation sequencing (NGS) libraries. For example, poly(A) RNA is isolated from RNA samples. This procedure is effective but laborious and does not allow for the characterization of long non-coding RNAs or other RNAs lacking poly-A tails. Furthermore, it is unsuitable for severely damaged samples, such as FFPE samples. Another method uses antisense DNA or RNA probes to hybridize unwanted RNA within RNA samples prior to NGS library construction. After hybridization, in one approach, the sample is digested with a double-stranded RNA-specific enzyme (RNAase H) to remove the RNA probes and unwanted RNA. However, this method is not very efficient and is full of technical uncertainties. In an alternative approach, the probe is a biotinylated probe, which allows for the selective removal of unwanted RNA from the sample by capturing probe/target RNA molecules on streptavidin-coated beads or surfaces. However, this method is time-consuming, costly, and only partially effective. Furthermore, bead binding and washing are difficult and generally result in significant sample loss due to non-specific binding and capture. The present disclosure provides a method, a blocking oligonucleotide, a composition, and a kit for depleting unwanted RNA species from an RNA sample. In one aspect, the present disclosure provides a method for inhibiting the synthesis of cDNA of one or more unwanted RNA species in an RNA sample during reverse transcription, comprising the following steps: (a) a step of providing an RNA sample comprising one or more desired RNA species and one or more unwanted RNA species, (b) annealing one or more blocking oligonucleotides into one or more regions of one or more unwanted RNA species within an RNA sample to produce a template mixture, and Herein, a step in which one or more blocking oligonucleotides bind complementarily and stably to one or more regions of one or more unwanted RNA species, and one or more blocking oligonucleotides include a 3' modification that prevents elongation, and (c) a step comprising incubating a template mixture with a reaction mixture comprising, under conditions sufficient to synthesize a cDNA molecule using one or more desired RNA species as template(s), wherein cDNA synthesis using one or more unwanted RNA species is inhibited: (i) at least one reverse transcriptase, (ii) one or more reverse transcription primers, and (iii) Reaction buffer. In another aspect, the present disclosure provides a set of blocking oligonucleotides that are complementary (preferably completely complementary) to a plurality of regions of an unwanted RNA species, wherein each blocking oligonucleotide comprises one or more modified nucleotides that increase its binding to a region of the unwanted RNA species. In a related aspect, the present disclosure provides a plurality of sets of blocking oligonucleotides. In another aspect, the present disclosure provides a kit for inhibiting the synthesis of cDNA of one or more unwanted RNA species in an RNA sample, comprising: (1) (a) one or more blocking oligonucleotides that are complementary to one or more regions of one or more unwanted RNA species in an RNA sample, each comprising one or more modified nucleotides that increase binding between one or more blocking oligonucleotides and one or more regions of one or more unwanted RNA species, or (b) a set or multiple sets of blocking oligonucleotides provided herein, and (2) Reverse transcriptase. In another aspect, the present disclosure provides a method for designing a blocking oligonucleotide to inhibit the synthesis of cDNA of one or more unwanted RNA species in an RNA sample during reverse transcription, comprising the following steps: (a) A step of generating multiple blocking oligonucleotides complementary to regions of one or more unwanted RNA species, (b) A step of filtering out unallowed blocking oligonucleotides, (c) a step of generating one or more groups of blocking oligonucleotid