3 replies to this topic

[Ahrenhase]

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?

[nofx]

To relieve any secondary structure in the RNA

[dvddecarvalho]

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

[molgen]

dvddecarvalho, on Apr 3 2009, 05:24 PM, said:

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.