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qRT-PCR ?contamination in RT- controls

qRT-PCR negative control

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#1 kjab

kjab

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Posted 21 April 2012 - 01:46 AM

Hi there all, I've just got a few questions on some qRT-PCR stuff. I'm pretty much a newbie at this so apologies in advance!

I'm optimising some primers in bacterial RNA, two housekeepers and three genes of interest. In each sample I use 2 RT+ 1 RT- negative control and 1 negative (water instead) blank. Machine is a Bio-Rad icycler and SYBR. Primers were made up freshly, plates prepared in a hood that was UV'd and DEPC water used. Filter tips used throughout.

I have used two sets of RNA, both extracted using Qiagen's RNeasy mini-kit. One was treated with a Qiagen on-column DNAse, the second was treated with both Qiagen on-column and Ambion off-column. cDNA was made with Qiagen's Reverse transcriptase set with a gDNA elimination step.

In my most recent run I found the following in both RNA sets;

First housekeeping gene (across a range of primer concentrations) the RT+ reactions amplified as expected, however the RT- amplified (Ct more than 10 cycles after the RT+) and the melt curve peak was different between the RT+ and RT-. The negative blank did not show any product

Second housekeeping gene (at a single primer concentration) RT+ amplified and RT- Ct was also around 10 cycles after RT+. However, on this one the melt curve peak was at an identical temperature

All GOI's RT+ amplified and on once again the RT- controls amplified 10 or so cycles afterwards. The melt curve peak was at the same temp.

These results were the same in both the different RNA samples.

I'm not sure what I can I conclude from this? Am I right in thinking that the identical melt curve peaks between RT+ and RT- suggest genomic DNA contamination? If that's the case, why should one of the primer sets have an RT- product with a different melt curve peak whereas the others had the same peak? I would have thought that an on and off column DNAse treatment would have been enough to eliminate gDNA?

Thanks so much in advance for any suggestions on what I should be doing.

Cheerio





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