I'd be very grateful for some ideas...
Blood plasma was generated by differential centrifugation (200g, 2000g, and 16.000g for 15 min). Frozen at -80deg for a few days. After thawing, plasma was filtered (0.8u) and around 2-4 ml of Plasma was diluted with PBS to around 15 ml (the UC tubes required a large volume), and centrifuged at 10.000g for 30 min, and the supernatant further for 120.000g for 2h. I saw a transparent very sticky pellet, which I resuspended in PBS (more or less, it was hard to) , then centrifuged for 1h. The pellet was hard to resususpend in Qiazol, so I heated to 37deg for 5 min, and it dissolved. However, after RNA extraction there is nothing on the bioanalyzer.
Hi Stefan,
It would be best to carry out all differential centrifugation steps in one go and with diluted plasma. Please dilute the plasma to 20% with PBS or HBSS. Centrifuge at 500 x g for 10 min; centrifuge resulting supernatant at 10,000 x g for 30 min; centrifuge resulting supernatant at 50,000 x g for 60 min; centrifuge resulting supernatant at 100,000 x g for 2 h.
Remove supernatant by inverting the bottle and blotting on paper towel (let the bottle stand in inverted position for 5 min). Now add 100-200 uL PBS and store the bottles at ~45 degrees angle such that the exosome pellet is completely covered with PBS. Let the bottles stand in this position overnight at 4C. Next morning you will notice the exosome pellet is nicely resuspended in the PBS. Please do not attempt to resuspende exosomes by pipeting up and down for it creates bubbles and a large quantity of exosomes is lost. Collect the exosomes and proceed for RNA extraction.
Good luck.
Hi Stefan,
It would be best to carry out all differential centrifugation steps in one go and with diluted plasma. Please dilute the plasma to 20% with PBS or HBSS. Centrifuge at 500 x g for 10 min; centrifuge resulting supernatant at 10,000 x g for 30 min; centrifuge resulting supernatant at 50,000 x g for 60 min; centrifuge resulting supernatant at 100,000 x g for 2 h.
Remove supernatant by inverting the bottle and blotting on paper towel (let the bottle stand in inverted position for 5 min). Now add 100-200 uL PBS and store the bottles at ~45 degrees angle such that the exosome pellet is completely covered with PBS. Let the bottles stand in this position overnight at 4C. Next morning you will notice the exosome pellet is nicely resuspended in the PBS. Please do not attempt to resuspende exosomes by pipeting up and down for it creates bubbles and a large quantity of exosomes is lost. Collect the exosomes and proceed for RNA extraction.
Good luck.
Hi Stefan,
I faced the same problem when I used exoquick exosome isolation kit. the final pellet was really hard to dissolve in Qiazol lysis buffer. However, you may noticed that it is not difficult to dissolve the final pellet in PBS.
After following the ultracentrifugation procedure, add 100-200 ul of pbs and mix gently until the pellet completely dissolved. After that, follow the protocol for isolation of RNA from liquid samples (plasma/serum).
Hope it will work you. good luck
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