RT product check
Posted 05 August 2004 - 04:36 AM
Posted 05 August 2004 - 08:12 AM
What primer do you use for RT, random hexmer or oligo dT? After RT, you can ran a gel and you will see a smear of cDNA of different size.
Do you PCR primers and PCR work? include a positive control with known good RT as the template. This is very important to troubleshoot the problem. The chances for RT failure are much less than PCR failure.
Posted 05 August 2004 - 03:33 PM
I agree with pcrman about your RNA: does it give you the correct pattern, good clean sample, no smearing, the top band being about 2X as intense as bottom?
Also true about pcr- does your positive control come up? Did this PCR ever work?
Did you run an RT control PCR on these samples? For example bActin or GAPDH, or another housekeeping gene. These should give you a product, so you know your RT worked