ficoll contamination??

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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ficoll contamination??

Postby selmir » Feb 24 2003 1:45 am

I'm trying to isolate RNA from blood, but samples on denaturing agarose gel appear somehow degraded and contaminated with positively charged molecules of high molecular weight. RNA yield is also poor. Any sugestions?? Lymphocytes are isolated by the ficoll-paque density gradient and RNA extracted by GITC lysis protocol.
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Postby jdnelson » Feb 24 2003 11:34 am

do you see this positive molecule in your marker lane as well? do you use ethidium bromide in your loading buffer? - cause it may be EtBr if the concentration is too high (sorry i can't find my reference on that at this moment). as for the degradation issue... pay extra attention to your workspace, make sure your reagents are fresh and RNase free and don't forget to change gloves often!.
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ficoll contamination

Postby kimq » Feb 26 2003 6:20 pm

A colleague obtained excellent RNA from blood using Qiagen's QIAamp RNA blood mini kit. it does not use ficoll.
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Postby Karl » Mar 07 2003 7:43 am

Try washing your cells up to three times in autoclaved PBS (diluted from a 10x stock with DEPC water if necessary) after gradient separation. If you use the Qiagen RNeasy it will work I tried it several times.
A more important factor: how old is the blood you use and did you freeze it before isolating? Try fresh unfrozen blood.
The gloves changing thing is more of a myth. Try to work as clean as possible and you don't even need gloves.

What do you mean by degraded and which yield seeems to be poor for you?
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Postby Meredith » Mar 10 2003 8:11 pm

I've been using the PAXgene kit from Qiagen to isolate RNA from murine blood. I get about 3 ug from about 200 ul of blood...I have no idea what a typical yield is SUPPOSED to be for mice, though. For human blood, you are supposed to get 4-20 ug per 2.5 ml sample (roughly). Mine looks good on a gel, although I don't check it every time since I don't have that much RNA to begin with. I plan on using the Agilent Bioanalyzer to check quality from now on.
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