reverse transcription 101 - how to do it from the basics (Sep/30/2007 )
I am new to the reverse transcription. Can anyone please post the protocol of a RT? Better yet, explain the purpose of each step. Thank you!
I step:Mononeuclear cell isolation on Ficoll-Hypaque density gradient
II step:RNA extraction by the guanidium phenol chloroform technique of Chomcyzinski and Sacchi (1987) or by RNA extraction Kit.
III step:cDNA synthesis:using Moloney Murine Leukemia Virus enhanced Reverse Transcriptase (Enhanced RT, Roche diagnostics,GmbH Germany) as per the recommendations of the manufacturer.
Fifty units of the Expand RT was used per ug of RNA. The cDNA synthesis was carried out in 20uL volume using 1ug of RNA.
IV step:RT reac.: take 1µg total RNA in a microcentrifuge tube and incubate at 650C for 10 min, and then placed on ice.
2. prepare Reaction master mix.
3. add 18ul master mix to each tube .
4. incubate at 300C for 10 min, then at 420c for 45 min and then put on ice and stored at -20oC till further use.
use 2μL of the cDNA (equivalent to 200 ng of starting RNA template) For carrying out PCR reactions,
Master mix , per reaction:
Distilled H2O =14.1μl
10x Buffer =2.5μl
Mgcl2 =1.5 μl
Primer F =1μl
Primer R =1μl
hmmmm it seems like maybe you're looking for a tutorial?
I found excellent help from Ambion's website; also from Promega's website and NEB's when I started doing RT reactions. I would browse their support literature -