Amplification in HK gene but not for target gene - (Jun/26/2014 )

Hello. I'm pretty new to real-time PCR so please bear with me. Extracted RNA from a couple of cell lines, reverse transcribed and undertook real-time PCR (Taqman) to assess gene expression levels. All RNA samples had good 28S:18S bands via agarose gels and some with a260/a280 of 2.0. However although I got amplification for my housekeeping gene, I didn't get anything for my target gene of interest. I've had a search around to troubleshoot but did not seem to come across anything apart from perhaps problems with the primer itself, or even incorrectly setting up the parameters on the real-time PCR machine. If anyone could shed any light on this would be greatly appreciated. Many thanks.

Did you run a gel with your qPCR products ? If so, did you get any bands for the sample of a wrong size (how big?) or no bands at all ?

Tabaluga on Thu Jun 26 18:52:00 2014 said:

Did you run a gel with your qPCR products ? If so, did you get any bands for the sample of a wrong size (how big?) or no bands at all ?

Thanks for the reply. No I didn't. That might be something to consider. Out of interest, what could be wrong?

If there are bands at the wrong size this means that that your primer did not amplify the correct target and bound to something else instead. If the unspecific bands are relatively small in size (less than 100 bp) this might indicate primer dimers.

Can you perhaps post the melt curve of your qPCR in the meantime ?