My transcript is not very long ( about 500 bp) but my first attempt to synthetize cDNA wiht total extracted RNA from biopsies failed to result in full CDS cDNA that I would need to perform sequencing thereafter. I used the Sensiscript RT kit from QIAGEN using oligo d(T) primers. Now I am wondering about some kit using a combination of oligo d(T) primers and random primers such as the BIORAD iScript cDNA synthesis or the QIAGEN QuantiTect Reverse Transcription kit or the Promega GoScript Reverse Transcription System...? What do you think about this new strategy for reverse transcription? Can you recommend me a kit in particular to reach out my objectives?
Hello Cecile
I usually use the Superscript IV RT kit from Qiagen that is very powerfull, especially when using then the Platinum Taq DNA polymerase High Fidelity to obtain cDNA.
If you know your transcript, why don't you use specific primers for the RT? When I have rare transcript, I use the 5' specific primer together with the oligodT, and it works!
Good luck!
Patricia
I have had some problems with creating cDNA of the RNase-Inhibitor from total RNA-extract from HeLa cells. We always get only fragments when using random or specific primers. The solution was optimizing the PCR reaction by adding DMSO. So maybe you don't need another kit, but using yours. Beside DMSO in the PCR mix, you can also try something like touch-down pcr. I think there are a lot of information on how to optimize PCR