Help! Smear in Real-Time PCR Product - (Jun/15/2011 )
I am trying to perform qPCR on cDNA samples from mice. I performed my cDNA synthesis with Superscript Vilo (Invitrogen), which is based on random priming. I have some samples in which I observe non-sigmoidal amplification plots, these samples have very low Ct counts (See attachment). And if I run these samples on an 1% agarose gel I observe smearing in addition to the PCR product of expected size (See attachment). For my qPCR I used 1:10 diluted cDNA with Quantitect SYBR Green Kit (Qiagen).
What are these smears on the gel, and how can I get rid of them?
Any ideas or suggestions or solutions will be greatly appreciated
It appears to me you either have genomic DNA contamination or RNA degradation. Can you run your RNA on an agarose gel to check both?