We use TRIzol® Reagent (Life Technologies) to extract RNA from heart tissues, cardiac fibroblasts, cardiomyocytes (mice, rats, pigs and humans). I'm a little concerned about the final step where it involves washing the pellet with ethanol. Even though we aspirate out all the ethanol, there are still droplets of it stuck to the walls of eppendorf tube. For downstream applications like RNA seq, I'm guessing ethanol will make a difference. In our case, we mostly perform qPCR with the isolated RNA.
I know that using a kit might lower the yield, purity of the extracted RNA. Can anyone suggest an alternative method or commercially available kit for isolating pure RNA?
Kindly help,
Thank you!
HI Jaya,
I used to have similar concerns so I purchased kimwipes that I could autoclave; I would gently dab out those droplets after the extraction.
You can also dry the pellet for up to 10 minutes on the bench, then tap out the drops.
However, you can use a Qiagen kit following (or instead of) Trizol, like the RNeasy Kit. Then you'll clean (or extract) your sample through a column and elute into a clean tube at the very end - no droplets. You do sacrifice a little yield, of course, but the purity is still good. There is also an RNeasy microkit for really small quantities of RNA; we've tested it in our lab and used it for RNAseq samples.
Good luck!
C
I tried dabbing like you said to get the droplets out. Sometimes the pellet is slimy and I'm concerned of loosing if I dab it.
Like you said, I will try using RNeasy kit. May I know the catalog number of RNeasy microkit you used?
Thank you very much for your response.
Hi Pratap,
I use the Qiagen kit, RNeasy Fibrous Tissue Mini Kit, Cat No./ID: 74704. I get a good yield and high purity, I have standardized the protocol for this kit though, and incorporated a couple of washing steps. I can send you the protocol.
Best,
Saisha.
Saisha Abhay Nalawade would you please send me your protocol for the RNA extraction of Fibrous Tissue of rat heart.
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