Extra peak in Electropherogram...Your opinion? - (Apr/28/2006 )

Hi All-

I have sent these images to two different tech support departments in two different companies, and I got different answers. For you RNA experts out there, can you please give me your opinion on these images..?

Sample 1 was isolated with a combined Trizol/Qiagen column protocol with an on-column DNase digestion. It is this sample that has the extra peak/band.

Sample 5 was isolated only with Trizol.

I was told by one that it is 45S RNA, a reflection of the cell cycle. I was told by another that it is contaminating gDNA. But if it is, would you not expect the contaminating gDNA to be present in the Trizol-only isolation - Sample 5 (no DNase or column purification)??

I have also attached a digital gel image...Samples 1-4 were isolated with the combined method and Samples 5-8 are Trizol only.

Thanks so much to those of you who take the time to check it out.

I would have thought that it is one of the rRNAs, not sure which one. Otherwise it could be fines from the column coming out in your elution step.

This is NOT NOT NOT gDNA. Anyone who thinks they could see gDNA as a peak on an electropherogram like this is, to put it bluntly, seriously misguided. What happens to an electropherogram when gDNA is present is that you get a broad peak that spans the whole size range and swamps the signal from the RNA

I have seen this before Pilar. My opinion is that it is the rRNA precursor, becuase you never see it when running cytosolic RNA

However that said, sometimes you see it and sometimes you don't. With lower concentration RNA you often don't see it, and that may be what iis happpening here. Also it should be noted that even slight contamination of your RNA with chemicals or proteins can seriously affect the way electropherogram runs, and your Trizol RNA is more likely to be contaminated than your "silica cleaned up/Trizol" RNA. To make sure this contaminant effect is minimised you should upgrade to the v3 software (most recent release)

As a validation, try normalising your RNA then running equal amounts on the chip, and compare the bands then

If you call agilent tech support email them the files and they will give you their opinion too. They are brilliant

To cut a long story short Pilar, don't worry about it. Just get on with your downstream

Hi,

I'd prefer the RNA from Trizol only. It looks much better.
If you look at the band below the 26S RNA, i.e. 18S RNA, there appeared to be "shadow" on top of the 18S RNA band as well. And the 5S RNA is missing (or reduced) from the combo-purified method when compared to Trizol only. Apart from that, there's smearing at the top part of 26S RNA.

I'd go for Trizol isolated RNA in this case.
Have you tried redigest with DNase to solve the gDNA or 46S RNA prob?

Cheers

Thank you John!!!!

I was absolutely convinced this was not gDNA! I had studied all the literature released by Agilent showing what gDNA contamination looked like, and this looked nothing like it. In addition, if I had such a serious and consistent gDNA problem, would it not show up in the Trizol-only samples? I have been arguing with the manufacturer for months as to whether or not this was gDNA. I tried all of their suggestions, including a 45-min DNase digestion, which made absolutely no difference in the presence or absence of the extra peak. It is such a relief to have someone who really knows what is going on. Thank you so much!

Oh, and I have put in a request with my technical lab services dept to upgrade the software. If that helps, it will be a huge weight off my shoulders.

MSGs,

I am also concerned with the shadows that show up on the 18S and 28S peaks. But I decided to go with column-purified RNA because the A260 readings of Trizol-only isolated RNA seemed greatly exaggerated. First, it looked like I was isolating 100-200 µg RNA from about 10^6 liver cells, which seemed incredibly high (don't you think?). In addition, the concentrations based on A260 were MUCH higher than those determined by the bioanalyzer, and the measurements between the two instruments did not correlate at all. I became convinced that I had phenol contamination in the samples. With column clean-up, the concentrations were similar and correlated well with one another. So, despite the clunkiness and time, I decided the columns were the way to go.

Thanks so much for your replies. This problem has been weighing on me for months!

Ok, so now I have another question (sorry if it is dumb..)..

IF the extra peak is precursor rRNA, and not an artifact of proteins or chemicals from the column:

Isn't the RNA from the Trizol-only sample, the one that you specified as cytosolic, the same as that which goes through the column? Essentially, why wouldn't the pre-cursor rRNA show up in the Trizol-only samples if it is in the column-purified ones? It seems as though the non-purified samples should have all the peaks, pre-cursors included, as well.

Thanks again!

Take a look at my previous post