total RNA extraction methods

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total RNA extraction methods

Postby nootropic » Sep 06 2012 3:20 am

Hi everybody,

I want to isolate E. coli tRNA to use it in a cell-free protein synthesis system. The approach is to isolate total RNA at first, following treatment with high-salt concentration (ammonium sulfate??) and stepwise precipitation with isopropanol to obtain the tRNA. For the isolation of total RNA I found two suitable procedures, one uses acid guanidinium thiocyanate / chloroform / phenol extraction (http://www.ncbi.nlm.nih.gov/pubmed/17406285) and the other uses only Tris-HCl-saturated phenol extraction (http://nar.oxfordjournals.org/content/28/12/e64.full).
Beside that guanidinium thiocyanate acts inhibitory towards DNase and RNase, what are the advantages of the Tris-Phenol method?
Which procedure is recommended in my case?

Thanks in advance,
cheers noo
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Re: total RNA extraction methods

Postby relaxin » Sep 06 2012 1:59 pm

For total RNA extraction, the guanidinium thiocyanate method is most popular and simplified method. You can use commercial Tri-Reagent (or Trizol), which contains a dye to help you with the phase separation.
I am not familiar with the column purification of tRNA in the second method. If you are not using the column purification, the guanidinium thiocyanate method will be better, since it removes most DNA.
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Re: total RNA extraction methods

Postby CrowSan » Sep 07 2012 2:58 am

We use Trizol (not affiliated) here quite a bit. Works well for mammalian cells. Contains phenol of course so make sure your lab has a disposal route set up (it should have).
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Re: total RNA extraction methods

Postby nootropic » Sep 09 2012 3:34 pm

Thanks for your answers, I see that I should have been more precisely with my second reference, where I was referring to the Procedure described within the "Bacterial cultures and extraction of total tRNA"-section.

"Extraction of cellular RNA was done following a reported procedure (Ehrenstein, G. (1965) Methods Enzymol., 12, 588-595) with minor modifications. Cells were resuspended in 1.5 vol of 1.0 Tris-HCl, 10 mM MgCl2, pH 7.2. Phenol (1.5 vol) saturated with the same solution was added and, after 1 h mixing at 4°C, the phases were separated by centrifugation. The aqueous phase was recovered and the nucleic acids precipitated with ethanol."

My question was more about the reason, why the extraction system used by Chomczynski & Sacchi contained guanidine thiocyanate, phenol and chloroform and why that from Cayana et al. in contrast contained only phenol.
Anyway, meanwhile I have performed further literature research and must say that it has never been so hard to find proper informations about a topic. On the one hand this was because of the fact, that to the time when the essential work on nucleic acid preparations or isolations was done, one used to say “soluble RNA” or “sRNA” and not “tRNA”, but which I used as central element within my search phrases. On the other hand, I encountered the problem that most of the articles about RNA extraction were describing a procedure for the use on non-bacterial (mostly mammalian) cells, mostly following the aim of mRNA extraction to perform transcriptional analysis, and could not be simply applied on E. coli cells with the aim to isolate tRNA for the use within a coupled in-vitro transcription/translation. But, while reading through the 21 papers I found about the topic, the whole question cleared up step by step and finally the puzzle was completed as follows:

Within the topic of nucleic acid isolation, one has to differ between further subtopics, respectively focusing on isolation of:
- Total DNA and RNA
- Total RNA without DNA, containing Insoluble, large-size RNA molecules and soluble, small ones (rRNA, mRNA)
- Soluble, small-size RNA molecules without large ones (tRNA)
- aminoacylated tRNAs
- Unmodified tRNA

To begin with the final result of my literature research: soluble, small-size RNA molecules / tRNA (sRNA) can be extracted directly from bacterial cells without the need of cell lysis just by the use of phenol as single extraction agent. This relies upon the action of phenol on cell membrane permeability, making it possible for small particles, like tRNA, to pass through, while mRNA, rRNA are already too big. If the interest lies upon the isolation of total RNA, containing ribosomal or mRNA, then acetone becomes integrated into the phenol extraction procedure for the reasons of altering membrane permeability to a point where the large-size RNA molecules can pass outside the cell.
(http://www.ncbi.nlm.nih.gov/pmc/article ... 2-0130.pdf)
(http://www.jbc.org/content/240/10/3979.full.pdf)
The sole use of phenol is limited to bacterial (or maybe yeast as well) cells. For extraction of nucleic acid from mammalian cells, it was necessary to develop procedures like the guanidine thiocyanate/phenol/chloroform-procedure due to the need of reagents for cell lysis, nuclease deactivation and antioxidatives.
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Re: total RNA extraction methods

Postby relaxin » Sep 10 2012 8:51 am

Phenol extraction is the original method for nucleic acid extraction, because it denatures proteins, including nucleases. The use of guanidium thiocyanate (a more powerful denaturant than phenol) was developed later for tissues with high level of RNase, such as pancareas. It became a popular reagent for RNA extraction for most tissues. Of course, if you are interested only in small RNAs, you should use the method developed for this purpose.
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Re: total RNA extraction methods

Postby nootropic » Sep 10 2012 2:10 pm

lol
couldn't you have told me that earlier? :lol:
(sarcasm, no criticism) ;)
Thanks anyway!
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Re: total RNA extraction methods

Postby relaxin » Sep 11 2012 8:52 am

nootropic wrote:couldn't you have told me that earlier? :lol:


Well, since I cannot tell your age, I have to play it safe. Some people could be insulted if I told them what we did in the good old days. :D
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Re: total RNA extraction methods

Postby flower » Dec 12 2012 11:29 pm

Hi nootropic,
I am currently dealing with the exam same topic: Isolation of tRNA without other RNA for a cell-free protein expression system.

First: Thank you very much for your post - you saved me a lot of research work!!! (I had the same problem, not finding literature)

Now my question:
a) Did the method (only adding phenol to E. coli cells) work? Would you specify your protocol?
b) How did you check the quality of your tRNA preparation?

I would be very grateful for an answer!
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Re: total RNA extraction methods

Postby nootropic » Dec 16 2012 1:08 am

Hi flower,
yes, the phenol extraction worked very well. I opened another thread where I presented my results because I was focusing specifically towards the isolation of tRNA and not for total RNA, here's the link: viewtopic.php?f=3&t=32413
The quality and quantity of the recovered fractions were assayed spectrophotometrically, regarding the 260/280 and 260/230 quotiens. I did this procedure three times now and each time, I got 2-3 mL of main tRNA-fraction (from 500 mL OD 3-4 culture on 2YTPG medium) with a concentration of pretty constantly 13 mg/mL tRNA with values of 2,0 +/- 0,1 for both quotients, what is pretty much as good as it could be and in every case absolutely suitable for in vitro experiments - what I can say because meanwhile I have my system running, using tRNA that has been extracted this way (and as well 5-formyl-tetrahydrofolate that has been cheaply synthesized instead of expensively bought ;) )
Apart from that, I am planning to make 2D denaturing urea PAGE of the tRNA fractions following ultrasensitive silver stain [1].
Keep in mind to resuspend your tRNA in pure ddH2O solo and in no case to use TE buffer (as adviced in most cases), because EDTA will kill your ribosomes, causing them to dissociate into their subunits.
What kind of system are you going to use? PANOx? Cytomim?



[1] Igloi (1983): A Silver Stain for the Detection of Nanogram Amounts of tRNA
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Re: total RNA extraction methods

Postby coralnerd » Dec 19 2012 9:09 pm

Hi all,

I'm having some trouble using Trizol to extract total RNA from a complex microbial community (rumen contents). The samples are to be used for Illumina sequencing for metatranscriptomics.

I've been getting inconsistent results where in some samples I get nice clean RNA, with two distinct rRNA bands on a gel, and no gDNA contamination, while in other samples (or even replicates of the same sample) I get no rRNA bands, or I have a lot of contamination with gDNA.

I've also found that performing a cleanup step after the Trizel extraction using the Qiagen RNeasy kit results in 3-5 fold losses of overall RNA quantity, although it does improve the A260/280 ratios somewhat.

Has anyone else experienced similar issues with complex microbial samples? Any suggestions on how to improve the consistency of the results?
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Re: total RNA extraction methods

Postby nootropic » Dec 22 2012 8:20 am

The composition of cellular RNA depends a lot on the growth phase, in which the cells were harvested. Try to passage some of your cell into new media and collect them at mid exponential phase. Pour the whole culture into a stirred glass beaker in an ice bath at first to cool the cells down as rapidly as possible and keep them on ice for further steps. Centrifuge at 2-4°C, freeze at -80°C, preferentially -196°C, store at -20°C for max. 3 months.
Also consider influence of the guanidinium thiocyanate in the trizol on your expected result. You might try to do a simple phenol extraction, as SCN, apart from being a denaturing agent, separates rRNA from ribosomal subunits. Chloroform is said to improve phase separation, but I never had problems with separation without it and couldn't find a lot of difference when I tested it one time.
It might be eventually indicated to optimize your precipitation procedure. Check for the use of 0.8 M LiCl, 2 M NaCl, 2-2.5 M NH4OAc or 0,3 M NaOAc for precipitation, as some have specific properties for several RNA-species.
You can definitely separate your nucleic acid mixture into the respective S-fractions by stepwise addition of IPA/EtOH and fractionated collection of the precipitates, see this link: viewtopic.php?f=3&t=32413
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Re: total RNA extraction methods

Postby nootropic » Mar 04 2013 11:06 pm

Ok, it's been a while that I wrote that, but I just noticed that the link to my writeup about tRNA extraction was incomplete. In case that anyone should still want to follow it, here's the correct one:

http://molecularbiology.forums.biotechniques.com/viewtopic.php?f=3&t=32413
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