Chloroform and isopropanol RNA extractions?

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Francesco Desiderio

Hi,

RNA extraction is particularly tricky due to the high possibililty of contamination. RNA, differently from DNA, has a simpler structure and related to this conformation it might get more difficult to extract purely. Isopropanol precipitation might get some "other" compounds, such as DNA.

Furthermore, adding chloroform to the TRIzol might determine lower quality of the RNA product. In conclusion, I would recommend you to try first the molecular biology grade if available.

For further information please check the links below:

https://www.thermofisher.com/order/catalog/product/10296010#/10296010

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/trizol_reagent.pdf

Best regards

Alejandro Martin

Molecular biology grade means that it has been certified to be free of nucleases and ribonucleases, and probably free of inhibitors (heavy metal traces, etc.) for polymerases. Given the amount of time and money you have probably spent in getting the samples, I'd say it is cost effective to buy the more expensive molecular biology grade reagents. It's not like using the cheaper stuff will not work -most of the time it does- but you might be playing russian roulette with your samples and your time.

Hope it helps!

Francesca L.

Alejandro Martin I agree, I was asking exactly for this reason, I don't want to waste my samples and I want to get high quality and quantity RNA. Do you maybe know if this product from SIGMA is ok: Reference: C2432 ?

Thank you for your help!

Francesca L. ,

Sorry, no idea. I have often been forced to use whatever chloroform bottle I can find in the lab to extract RNA, but this has been for not very demanding applications (isolation of a specific gene by RT-PCR, for instance). If I had the budget and delivery was fast enough, I'd certainly use molecular biology grade reagents.