I treated a purified RNA sample with DNase and also with RNase (separate). The DNase-treated prep looked identical to untreated (as expected), but the RNase-treated prep disappeared (expected) except for the generation of a new, large, intense band (unexpected).
The lanes are:
1) GeneRuler 1 kb Plus
2) Isolated RNA (original)
3) Overnight DNase digestion
4) Overnight RNase digestion
5 & 6) cDNA preparations
Note - this is not a denaturing gel, just 2% agarose.
Your band seems to be DNA of huge mass. You don't have it in your prep, I don't know how you have it only after RNAse digestion... Since DNA can not be self-created, there must be an explanation... Sounds crazy, but I would run on the gel the mixture of RNAse with buffer you used (suspect someone added by accident the DNA to it). To verify what is DNA/RNA on your gel add 10ul of RNAse A to the electrophoresis buffer and incubate the gel with agitation 1h. This enzyme will remove RNA from your gel. Keep fingers cross!
I agree with Jan,
However, first do a Phenol purification and then RNAse and DNAse treatment again.
It is dubious that RNAse is contaminated with DNA. I can see the bands of the same length after cDNA synthesis. Likely you did not use special revertase and cDNA conditions to synthesize so large cDNA so effectively. I am not sure that RNA fragments can form multimolecular complexes. But, who knows. RNA can perform so many functions like proteins. To check if it is RNA-multimers in the denaturing agarose gel (a number of protocols can be found in the Internet). In addition I can propose that this is complexes of RNA with protein. To check this stain your gel with silver (http://www.sciencedirect.com/science/article/pii/0003269783905997) or Coomassie Blue.
Seems like it can be gDNA... Did you put your samples on the gel in equal amount? It's possible that you can't see the gDNA contamination in original RNA sample cause of exhaustion of EtBr by RNA... After the RNA degradation the gDNA band became brighter...
You might want to treat your RNA prep with DNase or protease to rule out contaminating DNA or protein, respectively. if not DNA or protein, you might think of some "precipitated" chemical that also fluoresces.
If you used pancreatic RNase, then digestion will yield a variety of oligoribonucleotides ending in pyrimidine residues, quite possibly some rather large ones if the sequence is devoid of pyrimidines. Perhaps these bound non-specifically to multiple RNase molecules, resulting in a network with greatly retarded mobility. This is just a guess; the intensity of the fluorescence suggests that there is a lot of RNA up there.
I didn't see the posting on the RNaseA/T1 mixture; I'f have to say that the putative undigested fragment consisted of poly(A) which of course is an expected component of your digest, especially is it is poly(dT) selected RNA. But I'm beginning to buy into the oops suggestions that include DNA contaminating your RNAse prep or buffer.
RNase A/T1 mixture is used for gDNA cleanup, makes the story more exciting... Could you please run it with the buffer on the gel, since I'm nervous guy;)
Not sure about all your parameters. Just for fun, take some of the material and see what it looks like under TEM. How did you isolate this RNA?
Yes. Jan's control is a MUST. Run ONLY the RNAse in the gel. Very simple right??
If ONLY RNAse gives you a big band, then RNASe is binding EtBr or some other contaminant (DNA). In fact, you RNA may be completely gone and you band is all FROM PROTEIN RNASe !!!!
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