Heating RNA samples for electrophoresis - (Apr/03/2009 )
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?
To relieve any secondary structure in the RNA
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
dvddecarvalho on Apr 3 2009, 05:24 PM said:
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.