Antisense transcriptomes of cells

Patent metadata

Field	Value
Publication number	US-9637779-B2
Application number	US-200913131413-A
Country	US
Kind code	B2
Filing date	Dec 2, 2009
Priority date	Dec 4, 2008
Publication date	May 2, 2017
Grant date	May 2, 2017

Abstract

Transcription in mammalian cells can be assessed at a genome-wide level, but it has been difficult to reliably determine whether individual transcripts are derived from the Plus- or Minus-strands of chromosomes. This distinction can be critical for understanding the relationship between known transcripts (sense) and the complementary antisense transcripts that may regulate them. Here we describe a technique that can be used to (i) identify the DNA strand of origin for any particular RNA transcript and (ii) quantify the number of sense and antisense transcripts from expressed genes at a global level. We examined five different human cell types and in each case found evidence for antisense transcripts in 2900 to 6400 human genes. The distribution of antisense transcripts was distinct from that of sense transcripts, was non-random across the genome, and differed among cell types. Antisense transcripts thus appear to be a pervasive feature of human cells, suggesting that they are a fundamental component of gene regulation.

First claim

We claim:

1. A method, comprising the steps of:
isolating RNA;
treating the isolated RNA with bisulfite to convert Cytosine residues to Uracil residues, thereby forming bisulfite converted RNA;
reverse transcribing the bisulfite converted RNA to form cDNA;
determining nucleotide sequence of at least a portion of said cDNA;
comparing and identifying matches between (a) determined nucleotide sequence data of the cDNA and (b) sequence data of a strand of DNA, wherein the sequence data have been transformed in silico by changing Cytosine residues to Uracil or Thymidine residues; and
determining whether a determined nucleotide sequence was transcribed from the strand of DNA based on identified matches.

2. The method of claim 1 wherein the step of treating converts non-quantitatively Cytosine residues to Uracil residues, thereby forming incompletely bisulfite converted RNA.

3. The method of claim 1 wherein the step of treating converts at least 95% of Cytosine residues to Uracil residues.

4. The method of claim 1 , wherein:
said RNA is obtained by transcribing two complementary DNA strands;
said reverse transcribing is performed by synthesizing first strand DNA using bisulfite converted RNA as a template for reverse transcription; and
further comprising the steps of:
synthesizing second strand DNA using the first strand DNA as template; and
forming double-stranded DNA molecules from first and second strand DNA.

5. The method of claim 4 wherein the RNA is expressed from one of the two complementary DNA strands in a cell.

6. The method of claim 4 wherein the RNA is synthesized in vitro.

7. The method of claim 5 further comprising the step of removing ribosomal RNA (rRNA) from the expressed RNA to form rRNA-depleted RNA.

8. The method of claim 4 wherein the RNA is treated with bisulfite to convert at least 95% of Cytosine residues to Uracil residues.

9. The method of claim 4 further comprising determining the sequence of a double-stranded DNA molecule and comparing the sequence to a database of genomic sequence that has been transformed in silico by changing Cytosine residues to Uracil or Thymidine residues.

10. A method, comprising the steps of:
isolating RNA;
treating the isolated RNA with bisulfite to convert non-quantitatively Cytosine residues to Uracil residues, thereby forming incompletely bisulfite converted RNA having 95% or less converted Cytosine residues to Uracil residues;
reverse transcribing the incompletely bisulfite converted RNA to form cDNA;
determining nucleotide sequence of at least a portion of said cDNA;
comparing and identifying matches between (a) determined nucleotide sequence data of the cDNA and (b) sequence data of a strand of DNA, wherein the sequence data have been transformed in silico by changing Cytosine residues to Uracil or Thymidine residues; and
determining whether a determined nucleotide sequence was transcribed from the strand of DNA based on identified matches.

11. The method of claim 10 further comprising comparing (a) determined nucleotide sequence data of the cDNA to (c) sequence data of a strand of DNA, wherein the sequence data have been transformed in silico by changing Guanine residues to Adenine residues.

12. The method of claim 10 further comprising the steps of:
determining nucleotide sequence of at least a portion of said cDNA;
comparing and identifying matches or mismatches between (a) determined nucleotide sequence data of the cDNA; and (b) sequence data of a strand of DNA, wherein the sequence data have been transformed in silico by changing Cytosine residues to Uracil or Thymidine residues; (c) sequence data of a strand of DNA, wherein the sequence data have been transformed in silico by changing Guanine residues to Adenine residues; or (d) sequence data of a strand of DNA that has been derived experimentally from a biological source; and
identifying nucleotides which have been converted from Cytosine to Uracil in the RNA or identifying nucleotides which have not been converted from Cytosine to Uracil in the RNA based on the matches or mismatches.

13. The method of claim 10 wherein the isolated RNA is total RNA.

14. The method of claim 10 wherein the isolated RNA is rRNA-depleted RNA.

15. The method of claim 10 wherein the isolated RNA is polyA-selected RNA.

16. The method of claim 10 wherein the isolated RNA is size-selected.

17. The method of claim 10 wherein the isolated RNA is microRNA.

18. The method of claim 10 wherein the incompletely bisulfite converted RNA is reverse transcribed to form a first strand of DNA and the first strand is used as a template to form a second strand of DNA, which together form a double-stranded DNA.

19. The method of claim 10 wherein the RNA is expressed and isolated from a cell sample.

20. The method of claim 10 wherein the sequence data of a strand of DNA is in a database of human genomic sequence.

21. The method of claim 10 wherein the strand of DNA is a sense strand of a gene.

22. The method of claim 10 wherein the strand of DNA is an anti-sense strand of a gene.

23. The method of claim 10 wherein a match is identified when at least 32 out of 36 contiguous nucleic acid bases are identical between (a) and (b).

24. The method of claim 12 wherein a match is identified when at least 32 out of 36 contiguous nucleic acid bases are identical between (a) and (b) or (a) and (c).

25. The method of claim 18 wherein adaptors are ligated to each end of the double-stranded DNA.

26. The method of claim 18 wherein sequence from both ends of a double-stranded DNA molecule is determined.

27. The method of claim 11 further comprising tallying (a) individual determined nucleotide sequences which were transcribed from the strand of DNA separately from (b) individual determined nucleotide sequences transcribed from a strand of DNA which is complementary to the strand.

28. The method of claim 26 wherein sequences from both ends are separately matched to a database of genomic sequence and if the ends are separated in the genomic sequence by a length larger than a length of the double-stranded DNA molecule, identifying the double-stranded DNA molecule as derived from a spliced RNA molecule.

29. The method of claim 26 further comprising the step of determining that the sequences from both ends match to one gene only, match to the same gene, and match to the same strand of the gene in a genome.

30. The method of claim 29 wherein if at least two matched sequences in a bisulfite converted RNA molecule are discontinuous in the genome, identifying the bisulfite converted RNA molecule as a spliced RNA molecule.

31. The method of claim 18 wherein the first strand DNA is reverse transcribed utilizing random hexamers or random octamers as primers.

32. The method of claim 18 wherein the double-stranded DNA is size selected and used as a template for sequencing by synthesis.

33. The method of claim 32 wherein the double-stranded DNA is size-selected for between 120 and 200 bp.

34. The method of claim 18 wherein nucleotide sequence determination is performed using the double-stranded DNA as a template to synthesize DNA molecules.

Assignees

• Vogelstein Bert
• Kinzler Kenneth W
• He Yiping
• Velculescu Victor
• Papadopoulos Nickolas
• Univ Johns Hopkins

Inventors

• Vogelstein Bert
• Kinzler Kenneth W
• He Yiping
• Velculescu Victor
• Papadopoulos Nickolas

Classifications

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12Q2521/107

  RNA dependent DNA polymerase,(i.e. reverse transcriptase) · CPC title

• C12Q2523/125

  Bisulfite(s) · CPC title

• C12Q2539/113

  Differential Display Analysis [DDA] · CPC title

• G06F19/22

  Physics · mapped topic