I was isolating the exosomes from 40 mL of urine sample by ultracentrifugation method and further try to isolate total RNA from it for NGS. I was able to isolate total RNA conc. around approximately 5-9 ng/uL using mirVana kit. I followed the all precautions during isolation such as using ice all the time, and RNaseZap (decontamination solution). I also went for the RNA QC in which RIN NO was 1.5 due to which NGS was not possible. Please suggest me how will I improve my urinary exosomal RNA concentration or any alternative technique to do urinary exosomal microRNA profiling.
Hi,
first of all, don't trust RIN results. You can't use this method simply because your sample doesn't contain ribosomal RNA.
Second, using ultracentrifugation you loose a lot of exosomes. Try to use exosome precipitation kits.
Third, for precipitation of RNA use 80% ethanol, add Glycogen (to stimulate RNA precipitation), and incubate it at -20C for 1 hour.
And the last one, try RT-PCR instead of NGS. You hardly can find anyone who will run a good miRNAs NGS for your samples. No matter what they promise to you.
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