SINGLE-CELL MRNA QUANTIFICATION WITH REAL-TIME RT-PCR

• US 20100216194A1
• Filed: 10/22/2009
• Published: 08/26/2010
• Est. Priority Date: 05/03/2007
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG), Tataa Biocenter AB
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Stahlberg, Anders, Bergtsson, Martin, Kubista, Michael, Stroembom, Linda
• Granted Patent: US 8,642,266 B2
• Time in Patent Office: Days
• US Class Current: 435/91.210
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

The present invention is directed to a method for performing an RT-PCR for amplifying a target RNA comprising the steps of a) lysis of a cellular sample which is supposed to contain the target RNA with a lysis buffer comprising between 0.2 M and 1 M guanidine thiocyanate, b) diluting the sample to an extend such that guanidine thiocyanate is present in a concentration of about 30 to 50 mM, c) reverse transcribing in the presence of a mixture of first strand cDNA synthesis primers, the mixture consisting of oligo dT primers and random primers, and d) subjecting the sample to multiple cycles of a thermocycling protocol and monitoring amplification of the first strand cDNA in real time.

Claims

1. A method for performing an RT-PCR for amplifying a target RNA comprising the steps of

lysing a biological sample which is supposed to contain said target RNA with a lysis buffer comprising between 0.05 M and 1 M of a chaotropic agent,

diluting said sample to an extent such that said chaotropic agent is present for a subsequent reverse transcription step in a concentration of about 10 to 60 mM,

without any intermediate purification step, reverse transcribing said target RNA in the presence of a mixture of first strand cDNA synthesis primers into a first strand cDNA, said mixture consisting of primers hybridizing to a poly-A sequence and/or random primers, and

subjecting said sample to multiple cycles of a thermocycling protocol and monitoring amplification of said first strand cDNA in real time,

wherein said biological sample consists of not more than 100 cells, preferably not more than 10 cells and most preferably is a single cell.

2. The method according to claims 1 wherein said chaotropic agent is guanidine thiocyanate.

3. The method according to claim 1 wherein said lysis buffer comprises between about 0.2 and 0.5 M chaotropic agent.

4. The method according to claim 1 wherein during the reverse transcription step, said chaotropic agent is present at a concentration between about 30 to 50 mM.

5. The method according to claim 1 wherein the reverse transcription step is performed in the presence of 0.5 to 2% NP 40 (nonyl phenoxylpolyethoxylethanol).

6. The method according to claim 1 wherein the lysis step is performed for at least 5 minutes at a temperature between about 75°

C. to 85°

C.

7. The method according to claim 6 wherein the lysis step is performed further in the presence of proteinase K.

8. The method according to claim 1 wherein between the lysis and the dilution steps, the sample is frozen at temperatures between about −

20°

C. and −

80°

C.

9. The method according to claim 1 wherein said mixture of cDNA synthesis comprises primers hybridizing to a poly-A sequence and random primers.

10. The method according to claim 9, wherein primers hybridizing to a poly-A sequence and random primers are present in essentially equal molar amounts.

11. The method according to claim 9 wherein said primers hybridizing to a poly-A sequence and random primers are present in concentrations between 1 μ

M and 5 μ

M each.

12. The method according to claim 10 wherein said primers hybridizing to a poly-A sequence and random primers are present in concentrations of about 2.5 μ

M each.