Because i isolate 5 ml of culture media without serum and i got 1.2 ng miRNA . And i need 70 ng minimum that we can do PCR
Personally, I used 15ml in plates and 20ml in flasks then after 24hrs i followed with ultracentrifugation (ultra filtration can be used too) to exclude dead cells then proceeded with trizol extraction and also used miRNeasy and got good yield from both. It is unclear what size of plates you are using or type of extraction. Also, are the cells adherent or suspension? I worked on adherent culture so i had to swirl the media before aspiration.
I can't help with your exosome protocol, but I think you should try doing your PCR anyway. 1.2ng of isolated miRNA is actually quite a lot and I think your results might be ok :)
I'll go with Laura. Give it a try. How did you determine the amount of microRNA anyways?
We have been doing isolation from 300 µl cell supernatants isolated in 800 µl Trifast and got good readings for some circulating miRNAs. Obviously, results will be different after exosome isolation but not necessarily in terms of amount.
Best
Boris
Kindly check https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507751/
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