RNA extraction question for qRT-PCR

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Strosa8's picture
RNA extraction question for qRT-PCR

I am attempting to extract RNA from mouse tissues and eventually measure the expression of a few target genes.  I have never done an RNA extraction starting with a tissue sample.  What is the best method of extracting pure and clean RNA to make cDNA for qRT-PCR.


Ivan Delgado
Ivan Delgado's picture

In my experience, Trizol is one of the best reagents you can use to extract pure RNA. The main consideration is to homogenize your tissue well and fast, ideally under cold (4oC) conditions. For that purpose I used a glass dounce homogenizer.

Alternatively you can also use a commercial kit like Qiagen's RNeasy kit. The advantage of the kit is that it minimizes the chance that you will get protein, and other, contaminants if you prepare your RNA the "old fashioned" way. 

Good luck

Cythrawl10's picture
I find Trizol leaves to much

I find Trizol leaves to much impurities at the 260/230 wavelength, and you end up having to do an EtOH precipitation to clean it up.
To stabilize the RNA, resuspend your samples in RNA Protect from Qiagen and then homogenize them as Ivan suggests.
By far the best kit I have ever used for RNA extraction is Ambions mirVANA kit. You can simply eliminate the microRNA extraction step and just add the optional homogenizing step. I consistently get really high quantities of super Pure and intact RNA using this kit.

vanshita's picture
i agree with Ivan , TRI

i agree with Ivan , TRI REAGENT is the best method to extract RNA. u can homogenize ur tissue in 1ml of TRI reagent/mg of ur tissue. and then centifuge. u can get RNA ,DNA and protein separatly in ur tube.