Reverse transcription protocol

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Reverse transcription protocol

Pinciples:

Reverse transcription, the definition origined from the concept of reverse transcriptase. Reverse trsnacriptase was firsly found and isolated from reverse-transcribing viruses which use the enzyme to reverse-transcribe their RNA genomes into DNA, which is then integrated into the host genome and replicated along with it.

 

Materials:

RNA sample, reverse transcriptase, RNase H, dNTP, PCR tubes

 

Procedure:

1. Mix and briefly centrifuge each component before use;

2. Mix the following in a 200μl PCR tube:

RNA sample         (≦5μg total RNA)

Primer                                                                    1μl

(50μM oligo dT or 2μM gene-specific primer(GSP) or 50ng/μl random hexamers)

10mM dNTP mix                                                    1μl

DEPC-treated water                                            to 10μl

3. Icubate 5 min at 65°C, then put on ice for 1min;

4. Mix the cDNA synthesis solution in the indicated order:

10X RT buffer                                                         2μl

25mM MgCl2 4μl

0.1M DTT                                                                2μl

RNase Inhibitor                                                     40U

Reverse Transcriptase                                         200U

5. Combine 10μl cDNA mix with RNA sample and primers, mix gently, and briefly centrifuge.

For oligo (dT) or GSP, incubate 50min at 50°C

For random hexamer, incubate 10min at 25°C, then 50min at 50°C

6. Incubate mixture at 85°C for 5min, then chill on ice.

7. Briefly centrifuge, then add 1μl RNase H to each tube and incubate at 37°C for 20min

8. Store at -20°C or proceed to PCR immediately.