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Problem in RNA isolation, degraded, even though I pay extra attention to prevent - (Jan/30/2006 )

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I froze the tissue in liquid nitrogen and some of the tissue is quite long and some is quite fresh (1-2 weeks). And all my sample showed degradation.


a good thing to prevent RNA degradation is to have a quick lysis after thawing the tissue. I've never done on total tissues, but on cells, adding preheated trizol helps great to reduce RNA degradation (heating at 50°)
2 weeks frozen doesn't drive a degradation, and i think nitrogen snap freezing is a good process.


If you suspect that your initial tissue isolation and storage may be the problem, you might try this


I do it in the following way. It may help you as well.. when you take out the frozen tissue from nitrogen, weigh it quickly and put it into the lysis buffer (RLT with B-ME in QIAGEN Kit) before it thaws. I use glass and teflon homogenizer to homogenize the sample and i do it in ice.. then i transfer the lysate to QIA Shredder.. spin it at the max and then proceed further with the elute exactly as per given protocol in QIAGEN RNeasy mini kit.. Also wear gloves as your hand may contain a load of RNases. Make 70% ethanol in 0.1% DEPC treated water / or RNase free water. Elute RNA at the final step in RNase free water. If possible spray with RNase Buster (BD Biosciences).. also take utmost care while running gel that it's not contaminated with RNases.
Good Luck!


i have just two things to add.. always use double autoclaved RNAase and pyranase free tubes..and use prechilled spatulas or pestles for grinding etc...


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