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RNA integrity

RIN degradadation RNA TRIZOL Phenol

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5 replies to this topic

#1 mike2k

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Posted 21 August 2012 - 02:23 AM

Hi,

I´m new to this forum and I hope to find some help here.
I´ve recently started to isolate RNA from bacteria (spirochetes).

My RNA always looks kind of weird: Visualized in an "normal" agarose gel (but sample buffer containing formamide and formaldehyde) I see beautiful 16S and 23S bands. However, in the 5S region there is always a very big spot, which might correspond to more than one band. I have also run my samples through Agilents bioanalyzer - which can never generate a RIN - obviously because of this unexpected signal in the 5S region.

I assume this is degradation, isn´t it? What makes me think is that I currently use two different protocols (TRIZOL; Hot Phenol) - and the problem is with both methods. Another thing is that in the bioanalyzer if there was degradation I would expect a zick zack of the curve, which I don´t see. I have also to say, that I´m quite new to RNA isolation and that I´m not that experienced; but I´ve taken every precaution that I can think of to avoid RNAse contamination (gloves, RNAZAP, DEPC, extra pipettes etc..). Maybe I have overlooked something but I cannot think of anything.

I´ve uploaded an example from the bioanalyzer. If anybody could help me in interpreting this result I´d be very grateful!

thank you!

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#2 microfir

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Posted 21 August 2012 - 09:47 AM

Hi mike
I isolated RNA from various gram negative bacteria from all sort of physiological conditions (planktonic, biofilm, late stationery phase etc'), the only thing I can say for sure is that each bacteria has different "RNA pattern "(on gel and in biolanalyzer), some have more then one band (usually larger then 23S) some have different 16S:23S ratios.
Anyway your RNA doesn't look like it was degraded. It seems that part of your plot is lower then the base line (in the left part of the plot, right after the 23S peak). Maybe that’s the reason your cant get RIN value( ?)

Ofir

#3 mike2k

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Posted 22 August 2012 - 10:14 PM

Thank you for your answer!

I also want to believe, that my RNA is not degraded. However the inverted 16:23S ratio is strange. But what´s really puzzling me is the large peak in the 5S region. This is also clearly visible in an agarose gel. Sometimes it really looks like a double band, though it´s a smear more or less. What can it be? I think the tiny peak really is the 5S but the large peak right to the left of it, what is it? It can´t be tRNA, can it? And also for other small RNA the quantity seems to be too large....

I´ve checked the bioanalyzer data once more and I think the 5S region is the main reason why I cannot get a RIN number out of it. It says "unexpected signal in 5S region" with a white "X" in a red circle, which stands for "critical error" as I believe...

#4 mike2k

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Posted 04 September 2012 - 12:21 AM

I have now incubated some of my samples o/n @37°C to see if they become degraded. However, the signals nearly look the same (although flourescence intensities are a bit lower; so i guess there is some minor degradation going on, but nothing too serious). Thus, I think contamination with RNAses during the purification does not happen. I think it is maybe something with the cultures somewhere before or during lysis.

I´ve done now extractions with an E. coli culture in parallel (TRIZOL protocol) and they even look worse (however the 23:16S rRNA ratios are quite nice) - so it cannot be something with the species either. Can someone please tell me an exact protocol for RNA isolation of E.coli? (though, it´s a little embarrassing for me to ask this but maybe there is my mistake?) I took a culture of OD600 of 0.5 and took 1.5 ml. Pelleted them at RT 3´ 14 krpm in a microfuge. then removed s/n and added 1ml of TRIZOL - the rest is acc. to manufacturers protocol...

I´m happy for any answer,
thanks

Edited by mike2k, 04 September 2012 - 12:23 AM.


#5 cosiarz

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Posted 07 September 2012 - 10:02 AM

hi Mike!
are you sure you didn't overload your bioanalyzer? if you use 'pico' chip you might have saturated dynamic range

lukasz

#6 mike2k

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Posted 07 September 2012 - 11:53 PM

thank you! Yes, I´m sure. I´ve diluted the samples to 1-2 ng/µl and bioanalyzer confirmed this during the readings.

I´ve done now the hot phenol extraction with e.coli - there the mysterious 5s peak is nearly gone (however with my special bacteria it remains).. So I guess it must be something with the TRI method in this case. However, it must be something with the method per se, since I tried now compounds from different companies and they all look the same. So what can it be? I really have no longer logical explainations...And I also find it strange that it seems nobody else has this kind of problem. When I search the internet I cannot find anyody having the same issue...

by the way. does anybody know a good protocol for rna extraction from tissue? apart from the trizol method?





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