Iam using TRIazol-chloroform method for RNA extraction from PBMCs. However, the quality of isolated RNA is poor in some samples considering A260/280 ratios. are these samples valid again for downstream applications or should i just find other samples ? also, what should i do for future samples to avoid repetition of the same problems ?
Have you tried cleaning up the RNA after extraction? We use the Qiagen cleanup kit and it helps to improve quality
https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/rna-clean-up/rneasy-minelute-cleanup-kit/
If you don't have access to that, you can also try spinning your sample for a minute more before pipetting out the RNA in the aqueous phase and making sure to not go near the white interphase. You don't have to get all the aqueous phase out. See if only taking out half of it helps improve your ratios, if so, it's your pipetting that's getting protein contamination into your RNA.
M. C. Milfort thank you for your kind support.
would you recommend to repeat the chloroform step rather than increasing the spinning time ? or it wont make a big difference in both cases ?
also, iam expecting low RNA yeild form my sample, do you think its wise to take only half of the aqueous layer ?
thanks again
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