PFA Perfusion and RNA extraction

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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PFA Perfusion and RNA extraction

Postby newbie17425 » Feb 11 2016 7:50 am

Hi,

I am having some problems with RNA extraction from 4% PFA fixed rat tissue (hippocampus). I use the Zymo research RNA extraction kit but get a very low yield as low as 10 ng/uL. But when I do the same with a PBS perfused tissue, I get a good RNA yield (<700-800 ng/uL). Could you please suggest some ideas to improve the yield of RNA in 4% PFA fixed tissues.

Thanks
newbie17425
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Re: PFA Perfusion and RNA extraction

Postby relaxin » Feb 11 2016 11:00 am

Paraformaldehyde cross-links RNA with DNA and protein, making the RNA extraction difficult. You may try the kit below. I have used similar kit for DNA extraction, but not RNA.

https://www.qiagen.com/us/shop/sample-t ... nformation
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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Re: PFA Perfusion and RNA extraction

Postby newbie17425 » Feb 14 2016 12:33 pm

relaxin wrote:Paraformaldehyde cross-links RNA with DNA and protein, making the RNA extraction difficult. You may try the kit below. I have used similar kit for DNA extraction, but not RNA.

https://www.qiagen.com/us/shop/sample-t ... nformation


Thanks for the link "relaxin" and apologizes for my delayed response. I found similar products from Thermo Scientific as well. But the protocols is for using with tissue sections. I have whole tissue (eg - cerebellum). Will these product work with whole tissue also?

Thanks.
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Re: PFA Perfusion and RNA extraction

Postby relaxin » Feb 14 2016 4:12 pm

It should work. Just skip the step for the removal of paraffin wax.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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