RT product check - Can I qualify my cDNA? (Aug/05/2004 )
After a series of failed RT-pcR’s I’m going back and troubleshooting every step. The rna in question is showing up on a formaldehyde rna-check gel so I know the problem is with the rt or the pcr (using a two-step amplification kit) and I was wondering if anyone has experience with getting the cDNA to show up on an ag gel. I figure if I can convince myself that the RT product is fine then I can settle down and tinker with my primers or with my cycles\temperatures. Thanks guys!
What does your RNA gel look like, you can see 28s and 18s band without RNA degradation?
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.
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