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Does anyone have experience to extract RNA from frozen blood? - (Sep/30/2005 )

Does anyone have experience to extract RNA from frozen blood? The blood is kept in -80 for around 2 year.

-macrosky-

hi
i've not experienced on frozen blood directly, but on cells. I've noticed that i get far better results if i had the lysis buffer pre heated to 50° directly on the frozen cells pellet.
hope that may help you in your exp.

But what do you need particulary?
a colleague told me that for two-three month, -80° is sufficent to avoid degradations. But 2years is quite a long time for the tough RNA...

-fred_33-

I need to RT-PCR of an mutant mRNA from the patient blood. Since the blood is very precious, I don't want to waste any sample.


QUOTE (fred_33 @ Sep 30 2005, 11:09 AM)
hi
i've not experienced on frozen blood directly, but on cells. I've noticed that i get far better results if i had the lysis buffer pre heated to 50° directly on the frozen cells pellet.
hope that may help you in your exp.

But what do you need particulary?
a colleague told me that for two-three month, -80° is sufficent to avoid degradations. But 2years is quite a long time for the tough RNA...

-macrosky-

do you have only one sample or aliquots of the same one?

-fred_33-

QUOTE (macrosky @ Sep 30 2005, 10:28 AM)
Does anyone have experience to extract RNA from frozen blood? The blood is kept in -80 for around 2 year.

I have tried using Qiagen RNeasy kit on frozen blood. I added RLT to the frozen cells and tried to homogenize it. Since this is whole blood, there are abundant red cells and made it difficult to mix. I tried to homogenize it with a polytron and proceed with the rest of the purification. i did not get any yield. I believe that it may be possible to recover RNA if you used trizol instead of RLT. After the homogenization, add chloroform and centrifuge to remove the protein and heme which interfere with the binding of RNA to the column. Try this on a less precious sample first.

Andrew.

-andrewl-

Hi

I am currently having the same problem ie trying to get RNA put of frozen blood (sheep blood stored at -80C for over 2 months but less than a year). I have also tried RNeasy with no yield. I tried trizol and again no yield. My next step is to try trizol with acetic acid... someone has suggested that this may stabilise the cells but I am not sure. Will let you know if it works. I also tried RNAlater ICE as the product info suggests that it works with frozen cells but again the yield was not good... very low yield (6ng/µl) and on the Agilent Bioanalyser the samples just look degraded.

If anyone has tried any other methods I would be interested to hear how they turned out.

Auriol

-auriolw-

My advice...Don't even try, the RNA would be severely degraded after 2 years, even at -80.

Failing that. Spin the cells down. Grind under liquid nitrogen with pestle and mortar. The use the "Promega RNA isolation wizardy thing".

-Moz-

Most of the cells will be lysed already, so don't try and pellet the white cells or you will lose everything

Use Trizol BD on the whole blood sample. Pre-heat the trizol and add to the frozen blood. You will almost certainly need to add a chloroform step, and may need to go through the whole process twice, i.e. re-trizol your pellet

-John Buckels-