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Problems with RNA extraction or QPCR for AI

Problems with RNA extraction or QPCR for AI - Real-Time PCR and Quantitative PCR Forum

Problems with RNA extraction or QPCR for AI - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 03-10-2007, 05:23 AM
Pipette Filler
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Default Problems with RNA extraction or QPCR for AI



Can somebody help me,
When I test samples (alantoic fluid) on Avian Influenza, using QIAamp Viral RNA Kit for extraction and Taqman Influenza A/H5 detection kit version 1.0 for QPCR detection, I have no problem.
I am having problems with RNA extraction or Q-RT-PCR when I test samples that are inactivated with formaldehyde. The results are always negative, and when I test the same sample without inactivation with formaldehyde, results are positive. Where is the problem, in extraction or in Q-RT-PCR reaction?
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Old 03-24-2007, 03:58 PM
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Default Re: Problems with RNA extraction or QPCR for AI

Hi Deki,

you found the answer yourself: It is the formaldehyde. This is know to be a great inhibitor of your enzymes. That is why you kill you transcriptase and your polymerase and you do not get a signal in real-time PCR. The same is true for ethanol and other organic solvents.
My tip: Remove as much Formaldehyde from your RNA sample as possible. Even traces of FA will negatively affect your PCR reactions.
This is crucial and as you mentioned it does not happen with samples that are FA free. So wash wash wash that Formaldehyde out !!!!
Hope that solves the problem.
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Old 12-30-2007, 08:58 PM
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Default Re: Problems with RNA extraction or QPCR for AI

Hi Deki,
try other inactivation. I used many inactivated antigens, which are allontoic fluids inactivated and freeze dried (avian flu) and never had this problem!
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