I was extracting miRNA from lung cancer cell(A549) derived exosome by Total Exosome RNA & Protein Isolation Kit from Invitrogen™. I quantified the small RNA concentration with NanoDrop just after isolation. It was showing 1.59ng/ul and 1.29ng/ul respectively for two samples.
I think something is wrong with concentration. Can you please tell me about the normal range of exosomal miRNA concentration?
Is it possible to determine the miRNA concentration from exosome without RT-PCR?
I have to mention that pre-enrichment of exosome had been done by ultracentrifugation for 2 hours at 35000rpm.
It should be higher than you got! Be cautios at chloform-aquous phase extraction & confirm that all exosomes are compltely lysed. Another, tricky step is centrifugation, follow exact time & speed suggested by manual.
Exosomes have very low RNA amount, what you are getting is about right. Some folks perform qPCR "blindly"- without measuring RNA concentration, esp. when they process very low amount of sample (eg 100 ul serum) and they know that they have an extremely small amount of material, not enough for Qubit or Nanodrop. Others, who have more starting sample- just use maximal possible volume, to ensure they recover decent amount of exosomes and RNA
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