RNA isolation from frozen buffy coat

Sébastien Vigneau via methods%40net.bio.net (by sebastien.vigneau from gmail.com)
Tue Aug 17 11:53:26 EST 2010


Hi,

If the Trizol/tissue solution is too viscous, it may be that there is not
enough Trizol or too much tissue. The result of this will be that RNAse are
not efficiently extracted in the phenol phase or denatured by the guanidium
thiocyanate, which may result in RNA degradation. So, just increase the
Trizol/tissue ratio. In addition, don't forget to add glycogen in the Trizol
(1 µl of 20 mg/ml), which helps to get a good efficiency in the subsequent
precipitation.

I hope this helps,

Sébastien


On Tue, Aug 17, 2010 at 10:00 AM, Jovita Castelino <
J.M.Castelino from leeds.ac.uk> wrote:

> Dear Anita,
>
> My name is Jovita Castelino and I am a PhD student at the University of
> Leeds. I saw your message on the bio.net forum, which was posted some
> years ago:
>
> Hi,
>
> could someone give me some suggestions how to increase the yield when
> isolating total RNA from frozen buffy coat using the Trizol LS method?
> Now I add Trizol LS to the buffy coat even before I start thawing it
> because I am afraid RNases will work in the buffy coat. When thawed
> the buffy coat/Trizol LS solution is difficult to pipette, very
> viscous. I will be thankful for any help.
>
> Anita
>
>
> I wondered if you managed to solve the problem you had back then? If so,
> what did you do to get a better yield of RNA from frozen buffy coat? I am
> currently trying different kits to extract RNA from frozen buffy coat
> without much luck. I was thinking of trying out Trizol next but I want to
> make sure about it first.
>
> I would really appreicate any help/advice you could give me with this.
>
> Thank you,
>
> Jovita
>
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