I am working with the RNA isolation of mammary tissue. I have used TRIzol (500ul) and chloroform(200ul) to isolate the RNA. But after spin at 12,000g for 15min, I got a pink layer at the bottom and a upper white layer(I think they might be the fat). I got NO aqueous phase. After storing it at -80 overnight, I added another 100ul TRIzol and centrifuge for 15min, I got a upper aqueous phase and used the E.Z.N.A. HP Total RNA Kit to isolate the RNA. Then I used Qubit to detect the concentration of RNA(both broad range and high sensitivity), but the concentration of RNA is below 20 ng/ml. Does anyone know why this happened?
Hello
You need to use 0.2 mL chloroform per 1mL of TRIzol, but you did not. This will affect the quantity of aqueous phase for sure.
Any suggestions for RNA extraction of tissues with high chitin content? ... The top phase was removed and pellet was washed twice in Wash 2 and each time spin as ... 3) aspirate the PBMC layer(formed as a white ring below the plasma layer) ... I am looking for a proper protocol for RNA extraction from breast cancer tissue.
Hi Chunling,
The ratio trizol:chloroform should be kept as 1 of chloroform to 5 of trizol. Then you ll see a bigger aqueous phase.
Good luck!
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