I isolate RNA from plant via iRIS method, when i run it on denaturing gel ,i found no integral band, i made cDNA via Revet aid kit fermentas, when i checked it via PCR with House keeping gene with 50 bp ladder, an exact band of 160bp was get but i also found a band of 50bp, i dont know whether it is primer dimer or some amplified product. I also used ABS random primer kit , but got the same result. when checked in real time PCR , the Ct-value was coming at range of 25-27 which is not acceptable for house keeping, tell me what should i do.
cDNA preparation from degraded RNA
Posted 30 September 2013 - 05:59 AM
Do you see the 50bp product in your controls without primer? You have used this primer before with no additional bands or is this a recently designed primer? Does the appearance of the additional band only occur with this sample?
Posted 30 September 2013 - 08:52 AM
If you lack an intact band during the denaturing gel run, then your RNA has been degraded. Do you see a low-molecular weight smear? That's the telltale sign of RNA degradation. You might want to re-isolate your RNA.