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Problems with RNA extraction or QPCR for AI

Problems with RNA extraction or QPCR for AI - Basic Lab Protocols and Techniques

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  #1  
Old 03-10-2007, 05:39 AM
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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 fith formaldehyde, results are positive. Where is the problem, in extraction or in Q-RT-PCR reaction?
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Old 05-20-2007, 04:23 AM
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Default Re: Problems with RNA extraction or QPCR for AI

Hello Deki.

The Problem You Have Is Same For Me.

I Think The Inactivation Whit Formaldehydo
To Spoil The Core Of Genoma Viral And Estruture Of Rna....
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Old 07-27-2007, 02:13 AM
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Default Re: Problems with RNA extraction or QPCR for AI

Formaldehyde is a fixative, you've crosslinked your RNA (RNA-RNA, RNA-Protein). Carrying over formaldehyde to your PCR reaction will also crosslink your enzymes (protein). I've heard of reverse crosslinking with high salt incubation see [Only registered users see links. ]

Last edited by danfive; 07-27-2007 at 03:07 AM.
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Old 07-27-2007, 02:59 AM
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Default Re: Problems with RNA extraction or QPCR for AI

Quote:
Originally Posted by danfive View Post
Formaldehyde is a fixative, you've crosslinked your RNA. Carrying over formaldehyde to your PCR reaction will also crosslink your enzymes (protein). I've heard of reverse crosslinking with high salt incubation see [Only registered users see links. ]
See [Only registered users see links. ] for effects of formaldehyde on RNA.
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