Hi,
I extracted total cellular RNA from HEK293T cells by using a commercial kit. I left samples untreated or treated with 2ul RNaseA/T1 mixture (invitrogen) in the presence of PBS or protein lysis buffer NP40 (because I will use RNase in the presence of lysis buffer later). The labels of shared gel pic are as follows:
1) 1kb NEB DNA ladder
2) 500ng total RNA
3) 500ng total RNA incubated at 37C for 30min in the presence of water (untreated)
4) 500ng total RNA incubated at 37C for 30min in the presence of 1X PBS (treated)
5) 500ng total RNA incubated at 37C for 30min in the presence of lysis buffer (treated)
6) 100bp Promega Benchtop DNA ladder
1) Why there is only one thick band in 2 and 3 but no band or smear in 4? Why there is a band shift in 5?
I ran non-denaturing 1% agarose gel in TAE buffer at 30V.
my guess; 1,2= totally degraded, 3 you forgot to load the sample, 4= total cell RNA badly degraded (18 and 28 S ribosomal not visible) 6 RNA marker degraded or not denatured... but it is not clear not clear; what type of gel is it? denaturing? agarose? what kind of detection? radioactivity? ethidium bromide?
be very very carefull when you handle RNAse with RNA sample !!! (very easy to degrade the sample you do not want to be degraded!!!
for a quick RNA gel you should first heat your sample in at least 50% desioniezd formamide for 5 min at 80°C... then load on an agarose gel then color with ethidium bromide for total cell RNA you should see the 18 and 28s ribisomal RNA bands with nearly equal intensity.
Hi Didier, Let me add more information:
1) The sample was loaded in 3. I repeated 3 by using the buffer mentioned on invitrogen website, but the result was same. Nothing at all just like 3.
2) I don't have RNA ladder so I used DNA ladder.
3) This is a non-denaturing 1% agarose gel. I used "LabSafe Nucleic Acid Stain" from G-Biosciences which in non-radioactive.
4) I thought heating RNA samples in 50% form amide could be with denaturing gel. I am doing non-denaturing gel so I did not do that. Please correct me if I am wrong.
you have to denature your RNA samples to remove secondary structures even if your gel is not denaturing (it would be better to run a denaturing gel but it is much more work); for a quick control denaturing only your sample is fine.
for me your RNA is probably degraded, you should first focus on having a good total cell RNA prep then try your RNAse treatment on a prep you are confident with... after a RNAse A+T1 treatment you will only see a spot of very short RNA running on the bottom of the gel...
Thank you Didier. I will re extract the good quality RNA to validate the results. Although the quality of RNA was not good but can I still assume that absence of any visible band in 3 means the positive activity of RNase?
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