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Freeze thawing RNA


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#1 DNA

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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?

#2 pcrman

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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.

#3 noelmathur

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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.

#4 wntiong

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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.

#5 merlav

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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.
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I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie




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