upper ribosomal band missing!

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upper ribosomal band missing!

Postby lbbv » Oct 01 2007 10:51 am

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Hi there!!

here is a puzzled person working with plant RNA: my RNA samples SOMETIMES are showing the upper ribosomal band less intense than the lower one (in TBE buffer gels), and sometimes it is even missing! however:
- there are no signs of degradation
- the RNA performs absolutely fine in RT-PCR experiments.
- absorbance meassurements give 260/280 ratios of 1,9-2.2


It is not dependent on protocol (we use a special protocol since 11 years and it never failed untill now!), neither the samples (tried different pools), nor the buffers (tried different/new buffer solutions).

We've found some peole in the net with the same problem, but nobody seems to have an answer...

We need ultra-pure RNA for microarray experiments, and we need your help! Any suggestions/ideas are welcome!!

thanks for it!!

Nieves

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Postby r.rosati » Oct 01 2007 1:19 pm

Hm... It is kind of a common knowledge (that is, I can't point you to a paper) that the higher ribosomal band is the first to become weaker when RNA degrades, so that you can roughly guess the intactness of RNA by checking if the upper band is twofold more intense than the lower one.

Now, on plants, it could be something different... not sure...
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Postby lbbv » Oct 01 2007 1:30 pm

yep, I know...

but we see no degradation signs and, as I said, we can amplify a hosekeeping gene in a 1/4000 dilution. We've sythesised ds-cDNA and labelled it so it seems to be Ok, apart from the fact of missing a ribosomal band.

any ideas?
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Postby r.rosati » Oct 01 2007 2:47 pm

Hmm... are you briefly heating the RNA at 60°C to denatuere it, before electrophoresis?
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Postby lbbv » Oct 02 2007 11:28 am

Hi, thanks for the feedback!

yes, I've tried with and without heat-denaturation, cooling down to RT or placing on ice, and always the same: when the band is missing, it doesn't matter if heated or not.

nieves
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Postby lbbv » Oct 05 2007 12:02 pm

Hi,

I have done a test on the extracted RNA: I run the samples in TBE or in formaldehyde (denaturing) gels and the upper ribosomal band is missing in both gels. That means that the problem is not in the gel but in the sample.

As I have been using the RNA extraction method several years before, and it happens only to some samples, I am very confused.

any ideas????

Nieves
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Postby Carlton » Oct 13 2007 3:08 pm

Hi

I don't know how you are extracting your RNA or the species or tissue of origin, but I have read reports of people losing RNA in the cellular debris, especially the larger RNAs. The main culprits were thought to be the cell-wall fraction and difficulties in precipitating the RNA from the triazol reagent. If I remember rightly, they tracked the fate of the stable RNAs ie the ribosomes using C-14 uracil. I think the authors just went for a more destructive method of homogenizing the cells and avoided triazol.

Sorry if this a bit vague, I just saw your post and thought I'd put my two cents in. Interestingly, they were alerted to the problem when the 23S rRNA band (yes, it was bacterial) was of lower intensity, much like you describe.
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Postby yuvasoft » Oct 14 2007 10:11 am

nice article thanks for info :D
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Postby lbbv » Oct 14 2007 4:05 pm

Hi,

we are extracting the RNA grinding the tissues with morter and pestle. Our tissue is strawberry fruit (different ripening stages). We already considered that option and we grind our samples twice (anyway, we always used that system before and never had problems until now!!!).

As for the method it was published in 1991 and worked fantastic until now!!

Last extractions we made on monday last week (4 samples/2 replicates), only 2 samples were OK. We repeated on wendesday and we had other 2 RNA sampels OK (which had only one ribosomal band on Monday!).

And the FUNNIEST CASE EVER: from one pool, replicate 1 was Ok on Monday (replicate 2 had only one band) and viceversa: on Wednesday (replicate 1 had one band and replicate 2 had two ribosomal bands).

who understands this!!!!!?????

nieves
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Postby coddlecodd » Oct 15 2007 9:52 am

Usually a missing 28S peak is equal to degraded RNA. Since the 28S RNA is degraded much faster than the 18S and the size of your qPCR targets are generally smaller than the 28S you can still get decent qPCR results out of the RNA. Problem here is that you do, in fact, have degradation which gives uncertain values.
Do you have a difference in the Ct values when looking at samples with and without 28S peak?

There's also another possibility in that the missing 28S peak may be caused by salt contamination in the sample.

I've seen problems when extracting tissues with high salt or high lipid content regarding missing 28S band, my experience is that an initial Trizol step will get rid of this problem and protects the sample as well from RNase degradation, however complete homogenisation is crucial and if you can't do that in Trizol as mentioned here it's ofc not possible. I then continue with the aqueous phase to RNA column purification.

Cheers
/Joakim
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Postby lbbv » Oct 16 2007 10:12 am

thanks a lot for the ideas,

I must disagree with the degradation theory as I have seen the RNA samples in denaturing gels and I (as well as everyone who sees the gels) will bet my salary there is no degradation sign:
- I've seen degraded RNA before ant this is not the case
- no sings at the bottom of the lanes, even when/if overloading
- there is no smear from the lower ribosomal band

if it was something in the samples (lipids, carbohydrates or similar) I should have the same results in both replicates from same pooled material, which doesn't happen

our tissues doesn't take Trizol (or similar), neither any other standard kit (neither wonderful Qiagen kits) as we get nothing in the elutions or the tissue collapses any columm...

the protocol is as follows:
- grind tissue in liquid N2 and add tris/EDTA/Boric buffer
- phe/chl/IAA (neutral, pH 8), centrifuge and keep upper phase
- dilliute supernatants with water and sodium acetate
- add 0,4 volumes of 2-butoxyethanol (2BE), ice 30 min
- centrifuge and discard carbohydrate pellet
- add 0,6 volume of 2BE to the supernatants, ice 30 min
- centrifuge and keep nucleic acid pellet,
- ethanol washes
- resuspend in water and precipitate RNA with LiCL

the protocol works (well, worked) fine always and it is the only one I've found ot work OK in strawberry fruits.
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Postby r.rosati » Oct 16 2007 10:55 am

Given the protocol, I would suspect that some of the higher-MW RNA is lost during the carbohydrate removal step... Maybe you could try saving some of the upper phenolic phase in your next purification, and load it in parallel with your purified sample, just to see if the higher band is still there after phenol/chlorophorm.
You know, this reminds me of the alkaline miniprep lysis protocol, when you precipitate SDS with cell debris and high-MW DNA, while the small, supercoiled plasmids stay in solution. I'm really guessing here, but the way you add 2BE, how soon and how well you mix it afterwards could perhaps have an effect on the amount of RNA that's lost in the pellet.
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Postby mchlbrmn » Oct 16 2007 4:02 pm

Hi.
I'll toss this in for you to consider,although I don't really doubt that your lab's old/reliable protocol is probably fine. When I did a quick web search, the protocol that turned up (without needing to login for access) uses 0.4 and then 1.0 volumes 2BE, instead of the 0.4 and 0.6 volumes you use. Perhaps the additional 2BE makes the procedure more reliable at getting all the RNA down??

http://etd.lsu.edu/docs/available/etd-0 ... hi_dis.pdf
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Postby lbbv » Oct 17 2007 8:13 am

hi there,

it is a good idea to take aliquots to check where the upper ribosomla band dissapears, i'll do that next time. Although it doens't explain why it happens...

And for the protocol, it all comes from the original paper, which doesn't state it quite clear. But we all do the same:
- they add up to one volume (and previously had added 0,4 volumen)
- I said that we add 0,6 volumen (to the previously added 0,4 volume)

in the end, we all have a ration 1:1 (diluted extract:2BE). At the beginning, I tried all several combinations and this one was the one that worked better.
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Postby lbbv » Oct 17 2007 11:53 am

Hi, some news:

I made new RNA extractions (too late for the aliquots idea) and run the samples on normal TBE gels (I saw no differences when I run TBE or formaldehayde gels).

I quantified the samples and loaded equal amounts. It happens that:
- I have 4 out of 6 samples with double ribosomal band
- in those samples with only one ribosomal band, this one band shines stronger than the lower band of the sampel swith two ribosomla bands.

Does anybody knows if the upper ribosomal band can make some sort of structure which makes it run to the size of the smaller ribosomal band (remember that when I run the samples in denaturing conditions I see only one band...)??

I feel all this is an artefact of the RNA samples, but I can not demostrate it...

And thanks to all of you for your help!!!!!!

Nieves
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