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How to prevent RNA degradation in whole blood


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#1 wntiong

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Posted 19 March 2009 - 11:58 PM

any suggestion so that i can prevent as minimum RNA degradation in whole blood?

(for info, i will isolate RNA from whole blood, and i lysed my whole blood with Tri-reagent first, then stored in -80 before RNA extraction. On the way to transfer my whole blood to molecular lab, i transfer using ice pack and sealed inside ice box then immediately isolate RNA) {result: i got no pellet before and after 75% ethanol wash, and got a significant degraded RNA gel image}

which step went wrong? thanks

#2 Penguin

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Posted 20 March 2009 - 05:25 AM

Hi,
Are you doing all the RNA-safe things like using RNAse-free reagents, DEPC-treating your water, using ethanol from a bottle you know has been kept RNAse-free, always wearing gloves, using filter tips?? Usually doing all these things work for me but if you still have problems wipe down your bench/pipettes etc. with RNAse-ZAP (Ambion)

Ambion have a great page on handling RNA at http://www.ambion.co...trol/index.html

Hope this helps!!

P

#3 wntiong

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Posted 20 March 2009 - 06:37 AM

Hi,
Are you doing all the RNA-safe things like using RNAse-free reagents, DEPC-treating your water, using ethanol from a bottle you know has been kept RNAse-free, always wearing gloves, using filter tips?? Usually doing all these things work for me but if you still have problems wipe down your bench/pipettes etc. with RNAse-ZAP (Ambion)

Ambion have a great page on handling RNA at http://www.ambion.co...trol/index.html

Hope this helps!!

P


Hi Penguin, thanks for your reply.

I used all the RNA-safe things as you mention above. and our lab has a dedicated isolated work bench for RNA work only, and i used decon to wipe the benchtop everytime i start my RNA extraction. hmm... can it be the cause of RNAases in which i didn't autoclave my newly opened microfuge tube before i aliquot my blood samples (that going to isolate RNA from) into them?

#4 wntiong

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Posted 22 March 2009 - 03:56 PM

or perhaps, can it be freezer storage problem? i didn't store my samples into a closed lid microcentrifuge tube racks.




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