RNA isolation from PBMC in RLT-buffer

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RNA isolation from PBMC in RLT-buffer

Postby Sjors » Nov 06 2015 5:52 am

Hi everyone,

At the moment I need to isolate RNA from PBMC. The PBMC are in RLT-buffer and are in the -80 freezer for 3 years.
When I try to isolate mine PBMCs to get RNA, there are very low concentrations and no quality.
I tried to warm up mine samples in 37'C for 5 minutes and star mine protocol(wash with ethanol) at RT.
When i wash mine samples there is a white pulp (precipitate RNA/DNA?? Kristal’s) I think that is the problem of mine low concentrations and no quality at all.

Maybe you know how to handle this solution? How can I improve mine protocol to get good results of mine RNA isolation?
Has anybody also problems with RLT-buffer?
Does anyone know how long the quality of the RLT buffer is good in the freezer?

I hope you can help me!!!

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Re: RNA isolation from PBMC in RLT-buffer

Postby r.rosati » Nov 11 2015 1:03 pm

if I understand well RLT buffer is mostly guanidinium isothiocyanate, so I would say that it's possible to recover RNA from samples stored in this solution. There's at least one protocol on the web that says samples in RLT must be stored "for at least one year" (must've been "at most" one year). But even after three years, you might get some RNA.
But an important factor is whether the samples were completely homogenized in the buffer prior to freezing. When you thaw your samples, do they appear as a homogeneous solution, or do you still see the pellet floating around? If you can still see the pellet, then they haven't been disrupted in the buffer right away. Or they weren't mixed at all, since PBMC pellets basically disrupt just by thorough mixing in chaotropic solutions.
If you can see a pellet when thawing, then unfortunately you might have a hard time finding intact RNA.
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