2 replies to this topic

[Podge]

Hallo,

extracting RNA from human adipose tissue. Homogenized in Trizol with a PreCellys homogenizer. Then a standard RNA extraction including a centrifugation step after homogenisation to get the aqueous phase (no fats,proteins...). we also use Glycoblue.
Done it 2 times now on my own. Yesterday it worked perfectly. Had lovely blue pellets and when quantifieng spectrometrically I had high values. today I did the exact same. Same bench, same pipettes, same bag of tubes, same chemicals.... No nice pellets(sometimes something faint blue or something redish) and barely any RNA.
This leaves me really wondering what I have could done wrong, since it worked fine yesterday.

Any suggestions what I could be doing wrong?

Thanks for your help!

Edited by Podge, 29 July 2010 - 06:52 AM.

[fiat29]

What do you mean with blue pellet? I never heard about that. Just do usual trizol method without Glycoblue, It should be form white pellet, however keep in mind that after the pellet become dry, it seems like disappear, just continue with concentration measurement after add by RNAse free water.

[bob1]

How are your samples prepared (fresh? frozen (what temp)?, RNAlater?)?  Otherwise, you will have to review each step and repeat the extraction (which you will have to do anyway), as there isn't much that I can tell from what you have said.  Each step is important, so without a detailed method it will be very hard for us to suggest anything.

As you seem to have someone working with you - it might be easier to get them to watch you do the process so that they can see if you accidentally skipped a step or did something wrong - like added 70% ethanol instead of IPA during the precipitation.