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qPCR amplification plot suddenly not parallel.

qPCR real-time pcr mRNA cDNA

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#1 plin

plin

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Posted 16 July 2015 - 12:19 PM

Howdy all,

 

So lately I've beeing running qPCR to see if we can see changes in expression levels of our gene of interest "gene 1". All of these samples are derived from tissue.

 

I've attached a powerpoint showing the amplification plots from recent experiments, as well as one from a colleague who used the same primer on wt and knockdown tissue.

 

The weird thing we've been seeing is non-parallel amplification plots. This gene should be highly expressed in our tissue, and this primer has worked for years. When we ran the experiments back in June, and saw the non-parallel traces, we reordered the oligo and remade our primer mix from scratch and repeated the experiment on freshly isolated  tissue samples. We are only seeing these non parallel traces in our gene of interest, I've also included sample traces of our housekeeping gene (B-actin) and a second gene that had been probed in the same plate. We are not seeing the same oddities in these plots even though they are of samples derived from the same master mix.

 

We've personally had success with these primers in the past, and so we also looked at plots from a colleague who had used the primer somewhat recently. Her plots look totally fine, and you can clearly see the knockdown.

 

The other thing we see with our abnormal plots, is that the level of the plateau phase is unequal between the groups (much lower for some).

 

We use SYBR green, and 20ng cDNA per well. We use master mixes for the primer+SYBR and cDNA+water.

Can anyone offer any insight into what is going on? We're really not sure what is going on here.

 

Thanks,

Cynthia

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