is it degraded RNA? - I dont get a clear 3-band after electrophoresis (Jun/04/2011 )
hello everyone, I just used TRIzol to extract RNA from several cell lines. I followed exactly the protocol provided by invitrogen and finally dissolved RNA pellet with 150 ul DEPC-treated water. The OD260/280 ratio is around 1.95 and 260/230 around 1.1. Concentration ranged from 1.3 to 1.9 ug/ul. This seems ok I think. But after electrophoresis, I got a smear pattern instead of a clear 3-band pattern just as what I used to get. however, the cDNA generated from those RNA seems to give a similar pattern. Anyway, I put the figures here and hope you can tell me whats happening to my RNAs. Thanks.
I think your RNA is severely degraded, which is worse than your previous isolation. Following exactly the procedure in the protocol only cannot prevent RNase contamination. RNase is everywhere and can survive harsh conditions.
thank you, pcrman. I thought it was degraded too but the electrophoresis pattern is different from a typical one, like this one found on the internet. If it is really degraded, do you think if this could happen during the electrophoresis? Because I didnt use gel or buffer specific for RNA. The reason I am confused about the degradation is because that after performing RT-PCR today, I got specific band for GAPDH same as positive control, which means my RNA is fine?
Despite the pattern is different from what is shown in the gel (which is denaturing agarose gel) from Ambion site, there is no question that your RNA is degraded. You may also have DNA contamination in your RNA. Degradation of RNA (which is time and temperature dependent) during agarose gel run may not be a big concern, but still you should clean the comb and gel box with Rnase zap and RNase free reagents. The ability of amplifying highly expressed genes such as GAPDH doesn't exclude RNA degradation. When RNA is degraded partially, you may still be able to amplify highly expressed genes, but need more cycles.
pcrman on Sun Jun 5 22:34:14 2011 said:
Despite the pattern is different from what is shown in the gel (which is denaturing agarose gel) from Ambion site, there is no question that your RNA is degraded. You may also have DNA contamination in your RNA. Degradation of RNA (which is time and temperature dependent) during agarose gel run may not be a big concern, but still you should clean the comb and gel box with Rnase zap and RNase free reagents. The ability of amplifying highly expressed genes such as GAPDH doesn't exclude RNA degradation. When RNA is degraded partially, you may still be able to amplify highly expressed genes, but need more cycles.
Thanks. I used 1 ml of TRIzol for 107 cells, which is the limit according to the recommended protocol. If there is DNA contamination, do you think that is the reason? And I used -80 frozen cells, instead of fresh cells. Does it matter?
If GAPDH couldnt work, how about other non-housekeeping genes?
How should I test if the RNA is degraded or not? Even if it is degraded, the OD value is not affected, is that right? Is RNA electrophoresis the only way? Thank you very much.
I need to find out the reason for degradation because this is not the first time I came across this. I have been enough careful but it still happened.
after discussion with some labmate, I cleaned the tank, used new TBE buffer and tried electrophoresis with 2% gel at low voltage. Finally, I was able to see 28s and 18s band. So, the gel seems to be an important factor. Although I got more smearing background than my labmate did, I think it is due to high concentration of my RNA. Anyway, I will use this RNA to run qRT-PCR and see if it really work or not.