ReverseTranscriptionofRNA

實驗概要

The purpose of Reverse transcription of RNA is acquiring cDNA for follow research.

實驗原理

Reverse  Transcription (RT reaction) is a process in which single-stranded RNA  is reverse transcribed into complementary DNA (cDNA) by using total  cellular RNA or poly(A) RNA, a reverse transcriptase enzyme, a primer,  dNTPs and an RNase inhibitor. The resulting cDNA can be used in RT-PCR  reaction. RT reaction is also called first strand cDNA synthesis. Three  types of primers can be used for RT reaction: oligo (dT) primers, random  (hexamer) primers and gene specific primers with each having its pros  and cons. For a RT reaction, 1-2 micrograms of RNA is typically used.  Steps are:

1.         RNA is first incubated with a primer at 70 degree to denature RNA  secondary structure and then quickly chill on ice to let the primer  anneal to the RNA.

2.         Other components of RT are added to the reaction including dNTPs, RNase  inhibitor, reverse transcriptase and RT buffer.

3.        RT reaction is extended at 42 degree for 1 hr.

4.        Heat the reaction at 70 degree to inactivate the enzyme.

5.         Sometimes removal of the template RNA by treating the RT reaction with  RNase H is necessary before using the reaction in RT-PCR.

主要試劑

Random primers [ Details：from Promeg]

實驗材料

Total RNA 

實驗步驟

1. Prepare the following RNA/primer mixture in each tube:

     Total RNA                                               1-2μg

     Random primers (50 ng/μl)                       1 μl

     Heat 70℃ 5min, cool immediately on ice

2. Add the following components:

     5x RT buffer                                             5 μl                               

     10 mM dNTP mix                                    5 μl

     RHI                                                          1 μl

     M-MLV RT                                              1 μl

     DEPC H2O to final volumeTotal               25 μl

3. Incubate the samples at 37℃ (for Random primers, promeg) 60 min.

4. Prepare reaction master mixture. For each reaction:

     10x RT buffer                                           2 μl

    25 mM MgCl2                                          4 μl

    0.1 MDTT                                                2 μl

    RNAaseOUT                                            1 μl

5. Store the cDNA at -20℃ use for real-time PCR