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Heating RNA samples for electrophoresis


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3 replies to this topic

#1 Ahrenhase

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Posted 03 April 2009 - 04:33 AM

I'm running a native (non-denaturing) electrophoresis gel to see if there's any degredation in my samples. Most of the protocols I"m reading require that you heat the samples and ladders up (~70C) then chill them on ice. Is the purpose to denature any RNases that could be in the sample?

#2 nofx

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Posted 03 April 2009 - 05:47 AM

To relieve any secondary structure in the RNA

#3 dvddecarvalho

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Posted 03 April 2009 - 07:24 AM

To my knowledge, the best way to check RNA degradation is in a denaturing gel. This way you must see the two ribosomal bands (in the case of eukaryotic cells) and no smearing. In a native gel you will probably see some smearing but you can`t distinguish between degradation or RNA in secundary structure.
BTW, heating in this protocols is the denaturing step. RNases are not afected in such temperature.
I encourage you to do a formamide/formaldehyde denaturing gel.

Good look

#4 molgen

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Posted 05 April 2009 - 03:24 AM

To my knowledge, the best way to check RNA degradation is in a denaturing gel. This way you must see the two ribosomal bands (in the case of eukaryotic cells) and no smearing. In a native gel you will probably see some smearing but you can`t distinguish between degradation or RNA in secundary structure.
BTW, heating in this protocols is the denaturing step. RNases are not afected in such temperature.
I encourage you to do a formamide/formaldehyde denaturing gel.

Good look


I agree, the best way is on a denaturing gel. But, you can see the ribosomal bands very well on a native gel too.
I usually run a native gel and not a denaturing gel because of the formamide/formaldehyde. I try not to use them when possible.

If you see a smear on the native gel that is a back round then it's Okay as long as the ribosomal bands are sharp (the smear is your mRNA). If the ribosomal bands are smeared to then you have degradation.




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