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Are there anyone isolate exosome RNA by TriZol reagent?

I cannot see the PCR product and RNA pellets after i collected serum-free media (only DMEM) 48 h. What should i do and any other method for that.
asked Jan 7, 2015 in ExosomesTalk by BiggieTanapong

2 Answers

Yes, I have done this.  Here is what I did:

  • re-suspended the exosome pellet in 250 ul PBS, transferred to a 1.5 ml microtube, added 750 ul Trizol LS.  It's important to use Trizol LS, which is specially formulated for liquid samples.  
  • Performed the RNA isolation according to the manufacturer's instructions with one important modification.  After the phase separation, when you have transferred the clear upper aqueous phase to a new tube, add 5 ul GlycoBlue (available from Ambion). It contains glycogen, which can help precipitate smaller amounts of RNA. This amount of glycogen (7.5 ug) doesn't inhibit cDNA synthesis or PCR.  Also it contains a blue dye that makes it easier to see the pellet.  

This increased my yields x10.  Good luck!

answered Jan 24, 2015 by KristinL

Another tip:

In the same RNA precipitation phase cited by KristinL I place the aqueous phase with Isopropanol overnight at 4°C. I saw that can icrease critically the RNA yelds.

 

From: 

Exosome Isolation for Proteomic Analyses and RNA Profiling, Douglas D. Taylor, Wolfgang Zacharias, and Cicek Gercel-Taylor; Methods in Molecular Biology, vol. 728, DOI 10.1007/978-1-61779-068-3_15, © Springer Science+Business Media, LLC 2011

answered Mar 9, 2015 by MBonetti
edited Mar 9, 2015 by MBonetti
thank you for u comment
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