Problems with RNA extraction or QPCR for AI

[deki]

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?

[MARGARITA MARTINEZ]

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....

[danfive]

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 How do you reverse formaldehyde crosslinking?-DNA Methylation, Histone and Chromatin Study

[danfive]

Originally Posted by danfive

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 How do you reverse formaldehyde crosslinking?-DNA Methylation, Histone and Chromatin Study

See Effect of Fixatives and Tissue Processing on the Content and Integrity of Nucleic Acids -- Srinivasan et al. 161 (6): 1961 -- American Journal of Pathology for effects of formaldehyde on RNA.