problems with RNA bands - on agarose gel (Oct/30/2007 )
after i run as usual with agarose gel, i discover a weird things on my gel...
in fact, for RNA works, the gel shld only have the 18S and 28S ribosomal RNA bands and some lower molecular weight smear...
but for my stuff, i got a band above the 28S RNA band, around 1cm above from the gel picture...
at first, we thought it might be DNA contaminant, but after run with PCR, it doesnt show any product..
and when RNase A to treat it, the band disappear also...
so, we now in the doubt whether it is also the RNA band...
do u all ever face this kind of problem??
thanx for help...
in fact, for RNA works, the gel shld only have the 18S and 28S ribosomal RNA bands and some lower molecular weight smear...
but for my stuff, i got a band above the 28S RNA band, around 1cm above from the gel picture...
at first, we thought it might be DNA contaminant, but after run with PCR, it doesnt show any product..
and when RNase A to treat it, the band disappear also...
so, we now in the doubt whether it is also the RNA band...
do u all ever face this kind of problem??
thanx for help...
Hi Parami,
We had a similar situation, too. In our case, there were several different lines, some longer than 28S, some shorter. And we had several RNA samples, all extracted from the same type of cells in culture.
What we tried first was direct DNase treatment. You can try it as well, just take 3 samples. Treat one with RNase-free DNase, put only the DNase buffer to the second one and take the third without adding anything. Put all the tubes into incubation and run again in a clean (preferable RNase-free) gel.
We saw the same bands in DNase-treated samples as well, so we concluded that it may be an overexpressed transcript. If that's the case and you don't want to see it, maybe you can decrease the amount of total RNA you load into the gel. We typically load 400 - 500 ng and it's more than enough to check RNA quality.
Good luck
oh...
thanx...
never think of that problem before...
senior also got advise us before, load 0.5microlite is enough...
by the way, im goin to construction cDNA library after that...
is it ok if i just proceed with the step mRNA isolation, without treatment with DNase first??
and also, which brand is more suitable for mRNA isolation..??
promega or stratagene??
thanx...
in fact, for RNA works, the gel shld only have the 18S and 28S ribosomal RNA bands and some lower molecular weight smear...
but for my stuff, i got a band above the 28S RNA band, around 1cm above from the gel picture...
at first, we thought it might be DNA contaminant, but after run with PCR, it doesnt show any product..
and when RNase A to treat it, the band disappear also...
so, we now in the doubt whether it is also the RNA band...
do u all ever face this kind of problem??
thanx for help...
Hi Parami,
We had a similar situation, too. In our case, there were several different lines, some longer than 28S, some shorter. And we had several RNA samples, all extracted from the same type of cells in culture.
What we tried first was direct DNase treatment. You can try it as well, just take 3 samples. Treat one with RNase-free DNase, put only the DNase buffer to the second one and take the third without adding anything. Put all the tubes into incubation and run again in a clean (preferable RNase-free) gel.
We saw the same bands in DNase-treated samples as well, so we concluded that it may be an overexpressed transcript. If that's the case and you don't want to see it, maybe you can decrease the amount of total RNA you load into the gel. We typically load 400 - 500 ng and it's more than enough to check RNA quality.
Good luck