since htert rpe-1 are adherent cells , the only additional step I do is trypsinize pellet with dmem and then proceed with trizol lysis. Is this the step that's giving me problems in rna isolation? I have done rna isolation before with hek293t cells which doesn't need trypsiination for pelleting and I have got good results. Thought please ?
The best technique is to add Trizol directly to the plate or wells, after removing the media. Incubate the plate for up to 10 minutes at room temperature, and tip the plate from side to side to ensure the Trizol coats the entire bottom of the plate. The cells should lyse pretty quickly.
It's fine to trypsinize first and then add Trizol, but it's important the entire pellet is resuspended in Trizol. After centrifuging the cells, I remove most of the supernatant but leave about 100µL behind. I resuspend the cells in the 100uL of remaining supernatant, then I add 1 mL of Trizol. Then I mix either by pipetting up and down or by inverting the tube.
A common mistake is using too many cells with not enough Trizol. This means that RNases do not get completely denatured, and the end result is degraded RNA.
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