upper ribosomal band missing!

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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Postby coddlecodd » Oct 17 2007 9:36 am

Strange indeed, do you think LiCl can influence the peaks? What happens if you do longer or several washing steps after precipitation? I would really like to know why this happens from time to time and the only explanation that I have seen (bioanalyzer protocol for example) is that it's most likely salt if not degradation.
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Postby mchlbrmn » Oct 17 2007 10:28 am

You mean 2 bands becomes 1 band on a denaturing gel?
Now, bear in mind that I don't actually know what I'm talking about, because I'll be going off the wall...
Perhaps it's a something to do with the stain detecting the RNA. One form might more readily form secondary structure that can be stained and visualized after detnaturation, and the other one requires extended incuabation to start to form secondary structures?? Perhaps the time or amoung of mixing with high salt Li affects this?

You asked about a structure that would make an rRNA appear smaller. Well, wasn't it an rRNA that can splice itself in it's processing? Perhaps the big one cuts itself down to size? OK, never mind, I'm too far off that wall.
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Postby lbbv » Oct 17 2007 3:20 pm

strange isn't it? it is driving me crazy!

it is all very interesting but the problem is that I am in a hurry and I need the samples. At the moment I don't find a solution and the only thing I can do is to repeat and repeat the RNA extractions again and again until I get all the samples I need (as the problem happens randomly, I might be lucky and get all of them soon).

But RNA should behave and I know the protocol worked before (I used succesfully those buffers two weeks before everything started!!).

Another fact to consider is that I work in two different countries (Spain and Germany) and we have found the same troubles in both labs at the same time. The only thing in common is the material (but sometimes the RNA extractions work for that material)...

I wish I could show you the gels so you can be as certain as I am that the RNA is not degraded!

I've read some papers about ribosomal bands of high molecular weight bound by disulfure bonds that run like the lower ribosomal band (you see only one band in denaturing gels), but the problem here is that I KNOW that strawberry ribosomal bands have been normal until last month

I'll monitore the different steps in the extraction protocol and let you know soon!

thanks for everything!

Nieves
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Postby mchlbrmn » Oct 17 2007 3:35 pm

lbbv wrote:Another fact to consider is that I work in two different countries (Spain and Germany) and we have found the same troubles in both labs at the same time. The only thing in common is the material
...and sun spot activity??

Well, you're not studying rRNA, so screw it (?). Maybe you can just suppose it's possibly something to do with the conditions of RNA purification affecting the rRNA secondary structure, and use other means to ascertain the RNA qualilty. Were you useing it for RT-PCR? If so, then just try the samples out with several housekeeping genes, and if they work consistently then don't concern yourself with the exactly how the rRNA looks on a gel.
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Postby jop » Nov 09 2007 4:07 pm

I hope you have found a solution to your problem, but could it be the transfer of RNA would be less efficient for your large rRNA? Even if it worked before, maybe different batch of agarose, or buffer, and the transfer isn't as good?
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Postby lbbv » Nov 10 2007 6:48 am

Hi there,

it's been a long time without news from me, but that is only because things didn't change: we make RNA extractions and sometimes we get 8-out-of-8 right (that is with the upper ribosomal band more intense than the lower one) and next day, with the same reactives/buffers we find that 6-out-of-8 are showing the upper ribosomal band less intense (or even missing!) than the lower one.

AS I dopn't find the cause (and nobody seems to know how to fix the problem), at the moment I am repeating RNA extractions untill I get all the samples I need. At this moment, llant material is not a problem so I can repeat it as many times as needed. But the mistery is still there.

I have done RT-PCR experiments with several genes (and several amplification sizes) on those one-band-RNA and it seems OK: same amplification as with the good ones. That's why I keep thinking it could be an artefact but I can't find a way to find this out.

any help /suggestion is very welcome!!!

many thanks for all the answers and the feed back!!

Nieves
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Re: upper ribosomal band missing!

Postby hanes » Mar 23 2016 9:15 am

Hi there,

We are having the same problem. We analyse RNA samples on the Fragment Analyser and the upper ribosomal band is missing randumly in some samples.

If we reclean these samples by standard EtOH precipitation the 28s RNA peak reapears !

The RNA is definitely not degraded.

Salt contamination does affect all RNA peaks not just one. Tested and published by Agilent.
www.agilent.com/chem/labonachip Publication Number 5989-0712EN

Does any one knows what is going on ? what could interfere with the 28s RNA to make it undetectable ?

Hannes
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Re: upper ribosomal band missing!

Postby mdfenko » Mar 24 2016 7:03 am

it's possible that the samples which don't show 28s until they're recleaned have residual salts present.
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Re: upper ribosomal band missing!

Postby hanes » Apr 13 2016 8:37 am

Hi,

mdfenko wrote:it's possible that the samples which don't show 28s until they're recleaned have residual salts present.


If salt is a problem all bands should be affected this has been checked. It is not salt.
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