I'm trying to isolate RNA from suspended adipocyte now. The problem is that after centrifuging, the cells were still floating at the bottom, so when I remove all the media before adding RLT buffer, all the cells were gone. I tried to raise up the speed to 500g instead of the recommended 300g, but it didn't make any significant improvements. I also tried to centrifuge at 300g or 500g twice, but still no RNA could be detected after the whole process. Any idea what I could do to get RNA successfully? By the way, the RNA isolation from cultured cells was successful, so there should be nothing wrong with my RNA isolation technique.
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I could centrifuge my cells up to 1500g without damaging them (Human endothelial cells), maybe you can try triple your highest speed and see how it goes?
Also, How many cells do you have for each condition and what is the protocole you use for RNA extraction?
Try TRIzol Reagent for liquid samples -- TRIzol® LS.
We did it many times and each time successfully. Approved.
https://www.thermofisher.com/order/catalog/product/10296028
HI Henry! Thanks for your answer! I used fresh adipose tissue, cutting into small pieces and digested to remove RBC and connective tissues. Then I got preadipocytes in the media before RNA isolation--that's why I need to centrifuge all the cells down before lysing cells. The RNA isolation was done by QIAGEN RNeasy mini kit. The cell number was normal in the media before centrifuge compared to what we had before. After centrifuging it was not countable since all the cells were supposed to be precipitated at the bottom and then went directly to the lysis process.
For the speed you suggested, how did you know there was no damage on your cells? What protocols you used?
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