Freeze thawing RNA (View forum version)



DNA

Posted 17 March 2009 - 07:03 PM

Hi,

I was wondering to know after approximately how many times of freeze thaw cycle, the RNA goes off.
Do you have any idea? I thawed my RNA about 4 times and last time it did,t express well in xenopus oocytes and this time it did ,t express at all.
Do you think the RNA may have gone off?

pcrman

Posted 17 March 2009 - 10:10 PM

I don't think there is a golden stardard on how many times RNA should be frozen-thawed. The degradation rate dependents on many things such as RNase contamination, the method of thawing, the solution containing the RNA. Before use, It is better run a gel to check the quality if you are not so sure.

noelmathur

Posted 17 March 2009 - 11:10 PM

I second pcrman. I used to run my isolated RNA samples on Agilent Bioanalyser as soon as isolated, then depending on my needs, I would aliquot them. My rule was no thawing and freezing more than 3 times and so far so good.

wntiong

Posted 19 March 2009 - 12:42 AM

I second pcrman. I used to run my isolated RNA samples on Agilent Bioanalyser as soon as isolated, then depending on my needs, I would aliquot them. My rule was no thawing and freezing more than 3 times and so far so good.


Hi, i've similar problem here. I collected my blood samples from hospital, and there is a distance away from hospital to my uni molecular biology lab. So i stabilize my whole blood RNA with Tri-reagent (which is to prepare the RNA lysate first) and stored in -80C before i transfer to my lab after few days.

I will transfer the samples using ice box, drive for 30 mins and will probably isolate RNA immediately. However, i dunno how does this process (thawing during transfer, and freeze again in -80 while waiting for some preparation in lab) will affect my RNA.

I did an experiment last week, and isolated RNA using same method as above with my own blood. And, i got no RNA is seen on gel! can it be any processing or storage method above that i followed has gone wrong, thus make my RNA completely degraded?

Need help. Thanks.

merlav

Posted 19 March 2009 - 04:05 AM

If the RNA is to convert it to cDNA made the RT as soon as possible and convert all. So you don't have to worry to much about degradation.