Methods for preparing cDNA from low quantities of cells

• US 10,017,761 B2
• Filed: 12/23/2013
• Issued: 07/10/2018
• Est. Priority Date: 01/28/2013
• Status: Active Grant

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Weissman, Sherman M., Pan, Xinghua
• Primary Examiner(s): Bunker, Amy M
• Application Number:
  US14/139,612
• Publication Number:
  US 20140213485A1
• Time in Patent Office: 1,660 Days
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

Methods for preparing cDNA libraries from single and low quantities of cells are disclosed. The methods are based on the principles of multi-strand displacement amplification or semi-random primed polymerase chain reaction. The methods typically include a step of reverse transcription and subsequent amplification of cDNA. The methods can be adapted for preparation of cDNA libraries that are representative of mRNA or whole RNA expressed by the cell or cells. The cDNA is suitable for sequencing or microarray analysis.

Claims

1. A method of preparing cDNA from one or more cells, comprising sequentially

(a) preparing single stranded cDNA from cellular RNA of the one or more cells by a reverse transcription (RT) reaction comprising

(1) denaturing the cellular RNA;

(2) annealing one or more RT primers to the cellular RNA; and

(3) extending the RT primers to form the cDNA;

wherein the sequences of cellular RNA consist of RNA sequences from the one or more cells;

(b) preparing double stranded cDNA by second strand synthesis of the single stranded cDNA;

(c) circularizing the double stranded cDNA; and

(d) amplifying the circularized double stranded cDNA by a multiple displacement amplification (MDA) reaction comprising annealing one or more MDA primers to the cDNA and extending the MDA primers with a phi29 DNA polymerase to form a cDNA library representative of the mRNA transcriptome or whole RNA transcriptome of the one or more cells;

wherein steps (a) through (c) are carried out in the absence of active DNA endonuclease.

2. The method of claim 1 wherein the reverse transcription, the second strand synthesis, the circularization, the amplification, or any combination thereof are carried out substantially free from contamination of cellular genomic DNA.

3. The method of claim 2 wherein the one or more cells comprise a plasma membrane and a nucleus with a nuclear membrane, and the method further comprises preparing the cellular RNA for reverse transcription by lysing the one or more cells under conditions that disrupt the plasma membrane but leave the nuclear membrane intact.

4. The method of claim 2 further comprising substantially purifying the cellular RNA from the cellular genomic DNA prior to reverse transcription.

5. The method of claim 1 wherein the one or more RT primers are single stranded oligonucleotides selected from the group consisting of

(i) 5′

-phosphorylated oligo(dT);

(ii) 5′

-phosphorylated oligo(dT) with a 3′

anchor nucleotide that is not thymidine;

(iii) a mixture of random primers; and

(iv) any combination thereof.

6. The method of claim 1 wherein the MDA primer is a mixture of random primers.

7. The method of claim 1 wherein the whole MDA reaction is carried out for at least 8 hours at a temperature between about 26°

C. and about 40°

C. and in the presence of Tre [d-(+)-trehalose dehydrate] at a concentration of 0.2-1 M.

8. The method of claim 1 further comprising (e) fragmentation of the cDNA.

9. The method of claim 1 wherein the yield of the cDNA is at least 2 μ

g when the one or more cells is no greater than 5×

10⁵ cells.

10. The method of claim 1 further comprising ligating adaptor oligonucleotides to the amplified cDNA to facilitate sequencing.

11. The method of claim 3 further comprising substantially purifying the cellular RNA from the cellular genomic DNA prior to reverse transcription.

12. The method of claim 5 wherein the one or more RT primers are single stranded oligonucleotides comprising 5′

-phosphorylated oligo(dT) and a mixture of random primers.

13. The method of claim 8 further comprising ligating adaptor oligonucleotides to the fragmented cDNA to facilitate sequencing.

14. The method of claim 1, wherein the one or more cells are eukaryotic cells.

15. The method of claim 11, wherein the RNA is substantially purified from the cellular genomic DNA by harvesting cytoplasmic RNA from the cellular genomic DNA in the cell lysate.

16. The method of claim 2 comprising substantially isolating the cellular genomic DNA from the cellular RNA prior to reverse transcription.

17. The method of claim 1 wherein steps (a) through (c) do not comprise a template switching mechanism.

18. The method of claim 17, wherein step (c) is carried out under conditions that drive intramolecular circularization and reduces linear concatemers.

19. The method of claim 1, wherein the cDNA library comprises a uniform representation of the full-length cellular RNA expressed by the one or more cells.

20. The method of claim 1, wherein the one or more cells is less than 100,000 cells.