I have frozen blood samples - preserved in RNAlater stored at - 80. I'm planning to use the Ambion Ribopure blood kit or Trizol for RNA isolation. Can anyone provide me a protocol for removal of RNAlater from the samples - thawing, centrifugation, etc? Thanks.
If you will try to centrifuge your unfrozen samples you will lose a lot of RNA with destroyed cells. There is some protocols, like RiboPure RNA purification (blood) Kit, which can be used with RNAlater treated blood directly
@Timur Yergaliyev : I will be using the Ribopure blood kit for isolation of RNA. However it wasn't possible for me to do fresh extractions, and hence these samples were stored. I just wanted to know the protocol for removing RNAlater from those samples before proceeding for extraction with Ribopure or Trizol. Thanks.
Hello, Ankita Bane,
I would recommend to thaw your frozen samples in ice bath (0 - 4°C) and then spin in precooled centrifuge (4°C), 2000 g, 10 min, to harvest pellet. Then discard supernatant (optionally wash by some buffer (PBS) and repeat centrifugation) and proceed with isolation of RNA, e. g. by TRIZOL.
Please, let me know, if it works
Martin
@ Martin,
I use a similar protocol for removal of RNA except the washes with PBS. The pellet size in that case is extremely big and hence requires more amount of lysis solution. I'm guessing some amount of RBCs would get washed off with PBS. Thank you for the suggestion. I will let you know how it goes.