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rna degradation - what goes wrong

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#1 wntiong



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Posted 29 September 2009 - 12:48 AM

after running in 1% gel, my samples all appeared degradation (long smear with no 28 and 18s bands at all). I wonder what goes wrong since i quite confident with my skill and the protocol after few months of trying.

I keep my whole blood sample in tri-reagent in -80, and only isolate rna after one or two months (the max - because lab is too far away from the collection centre). I transferred my samples with ice bag and drove a journey of approx 30 mins, and isolate immediately. Then follow every single step as Tri-reagent protocol required, except that after isopropanol precipitation, i transferred and pool the pellet from aliquots (of same sample) into one tube then only do ethanol washing. This helped me to improve my rna yield. I air dried my pellet and if got remaining ethanol i use pipettor to remove it. Then stored the rna with rnaase-free water in -80c.

#2 Adrian K

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Posted 03 October 2009 - 12:41 AM

You mention you keep your sample for 1 months before you extract it.
Why don't you extract your sample straight away and then keep it in -80C and I'm sure your product will got reduced smear, and try not to repeat pipette.
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#3 noelmathur



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Posted 09 October 2009 - 05:57 AM

1. Check your gel tank, if its also used for running DNAs.
2. Check the buffer that you use. Use autoclaved water for making buffers. (I really don't know if you can use DEPC-treated water for making buffers so there is no trance of RNAse while running a gel.
3. Instead of running a gel, use Agilent Bioanalyser 2100 if you have access.

4. Carry your material in dry ice rather than plain ice. I would carry at highest temperature of -20C.

Otherwise I don't see any issue.

Edited by noelmathur, 09 October 2009 - 05:57 AM.

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