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qPCR vs Northern Blot

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qPCR vs Northern Blot

Postby Nevthonien » Thu Nov 07, 2013 8:35 pm

Hello everyone,
I have a question regarding comparison of results from northern blot and qPCR.
I have RNA isolated from samples in which my GOI should be silenced (samples from stable cell line, silencing by shRNA). Firstly I tried to check if silencing works by northern blot. It looked that my silencing has around 80% efficiency. Later I wanted to confirm this result by qPCR - and then surprise - no silencing (or max around 20% efficiency). Primers had efficiency checked, and qPCR had no contamination, melting curve looked fine etc. Then I ask you - which result should I believe? Of course I'm going to repeat this, so far I just did technical repetition of qPCR (no change in results), but all hint would be warmly welcomed :).
Best regards,
Netvhonien
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Re: qPCR vs Northern Blot

Postby jonmoulton » Thu Nov 07, 2013 10:12 pm

I assume you are assaying aliquots of the same RNA samples by the two methods. Does your Northern probe bind between the binding sites of your PCR primers? If not, it is conceivable that a change in splicing in the presence of the shRNA causes your Northern target to be spliced out of the pre-mRNA while the binding sites of the PCR primers remain. This would match your observations of a decrease in the Northern signal but maintenance of the RT-PCR signal. However, if the Northern probe binding target lies between your RT-PCR primer binding sites, that is an unlikely explanation of your observations.
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Postby JackBean » Mon Nov 11, 2013 1:28 pm

Are your primers designed to bind on two different exons?

@jonmoulton: if it was just partially degraded, there would be change in size of the bound RNA
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: qPCR vs Northern Blot

Postby jonmoulton » Mon Nov 11, 2013 4:38 pm

I see that I reversed the sense of the observations - the Northern signal was maintained, the PRC signal decreased. A splicing change could still produce that result, as could the RNA degradation Jack mentioned.
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Re: qPCR vs Northern Blot

Postby Nevthonien » Tue Nov 12, 2013 2:47 pm

Thank you both for answers.
Firstly - aliquots for Northern blot and qPCR were the same (firstly I did NB and after a couple of weeks qPCR).
@jonmoulton - you're rigth first - in Northern blot I had silencing, in qPCR i didn't. As far as your question is concerned - my NB probe covers whole length of mRNA - all exons (it's a short one ~2 kbp total). In case of my PCR primers - FORWARD primer binds in one exon in the middle of mRNA, and REVERSE binds at exon-exon junction of the next exon.
After strange qPCR result I checked one more time integrity of my RNA aliquot by electrophoresis and it looked ok - so I don't if it's a problem with degradation???
Any replies will be much appreciated.
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