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Real time PCR for degraded RNA


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#1 Mohamed 1984

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Posted 06 May 2014 - 11:58 AM

Dear All, 

 

I have RNA with an RIN of 3-5 meaning a bite degraded RNA. I am afraid to perform qRT-PCR for the cDNA of this sample if my primer did not amplify the product.

I designed primer that amplify 304 nt product for the HA of influenza virus whose length is 1717 nt.

 

How can I be sure that my primer will work perfectly.

 

Thanks  



#2 Trof

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Posted 06 May 2014 - 12:38 PM

You can't.

 

If you know you have a bad quality RNA, you may use as small assay as possible (below or just around 100 bp), I have some assays as small as 70bp.

But qPCR with degraded RNA be always a lottery. You have no guarantee that your target RNA is degraded preferentially, so what you measure is not how abundant the RNA really was. But many people use that, because they just can't get better.

 

However RIN number only asses ratio of rRNAs, which is supposed to express the degradation within the whole sample. But some organisms have different ratios, that expected, also if you have a non-cell material, you will only have a traces of rRNA there.

Is your RNA from cells or a non-cell? (also possibly if those cells were dying.. on a virus.. they may also have skewed RIN, and yet the the viral RNA may be intact)

 

So, first question is whether you really have a degraded RNA or not.


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