Isolation of total RNA from Escherichia coli :

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Isolation of total RNA from Escherichia coli

Modified on: Wed, 11 Apr, 2012 at 4:43 am

The following protocol for RNA isolation from Escherichia coli is from the OpenWetWare Wiki at
http://openwetware.org/wiki/Sauer:RNA_Purification_from_E._coli
(http://openwetware.org/wiki/Sauer:RNA_Purification_from_E._coli)

My Experience Purifying RNA from E. coli

Regarding RNA extraction, there is a horrible tendency of people to use kits for RNA extraction with bacteria. With
Qiagen (anion exchange) or other silica-binding technologies, the smaller RNAs are not recovered. In addition, cell
lysis is completely variable. This led me to find methods of extraction that recovered the RNAs in the same proportions
they are found in the cell.
I first noticed that the ratio of 16S and 23S to 5S (and tmRNA) was WAY too low in RNA prep'd by resuspending E. coli
in tri-reagent (called Trizol from here on) as the "lysis" step. Even though Trizol has phenol and guanidinium
thiocyanate, and the samples were heated to 65 degrees for 30 minutes, the large RNAs just didn't get out (a concern
for anyone doing quantitative mRNA Northerns as well).
So, I did several things. This lead to my current protocols.
For some experiments I was doing, I had chased 14C-uracil into a growing culture for several generations to label
stable RNAs. This was a good reagent to compare the losses of radioactivity with different procedures. 32P-PO4 is no
good for this type of experiment, it labels all sorts of stuff. For all of my experiments, I use an RNase I- (rna-) strain to
prevent this voracious periplasmic RNase from ruining my stable RNAs during lysis. If you plan on working with RNA in
E. coli, I recommend knocking at least this gene out. It has no known phenotype.
Keeping in mind that with long chases, radiolabeled substrate pools in the most stable material, so I was primarily
looking at ribosomal RNA extraction and recovery. As I suspected, straight into Trizol only recovered about 20-30% of
the counts in the soluble supernatant. As mentioned above, this was primarily loss of large rRNAs.
I tried resuspending the cells in trizol, and using zirconium beads in a disruptor (not that crappy one on a vortexer, but
a special machine that obliterates the sample). This busted the carcasses and liberated most of the counts. It told me
that the cell wall carcass was probably the problem.
I then prep'd the cells by completely lysing in B-PER (a commercial lysis detergent) under native conditions with egg
lysozyme and magnesium present (to keep the ribosomes from falling apart during lysis), then I added Trizol. This
does a good job at liberating practically all of the radioactivity from the insoluble debris; however, I found that
precipitations from Trizol (the next step), even with a precipitation carrier, only recovered about half of the RNA!
I no longer use Trizol.
A comparison of the RNAs from the above procedures showed that the recovered amounts of the small tmRNA and 5S
rRNA per cell was similar in each case, and that the large rRNAs were reduced in the Trizol-only samples.

My Current RNA isolation protocol for E. coli

For unstable RNAs like mRNAs

http://dualsystems.freshdesk.com/support/articles/8238-isolation-of-total-rna-from-escherichia

15/10/2014

Isolation of total RNA from Escherichia coli :

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Immediately stabilize the RNA, I like to add one volume of culture to one volume of cold methanol in tubes sitting on
ice. Then I add beads and mechanically disrupt the cells. I have also used "RNA later" and similar stabilization
reagents. Then I proceed to the organic extraction described down below.

For stable RNAs

I dilute the cells into an ice-cold salty washing buffer (seems to really help lysis) 10 mM Tris-Cl pH 8.0, 150 mM NaCl.
Then harvest in the cold. Resuspend the cells in a lysis buffer such as the one that follows. The Mg++ is there to keep
ribonucleoprotein assemblies together and to let the DNase work during lysis.
Native Lysis: B-Per (probably optional, but I have it on hand) supplement with 1 mM Mg-OAc, 0.5 mM CaCl, ~0.1 mM
EDTA, 0.01-0.1 mg/mL lysozyme, and RNase-free DNase (I use Roche DNase I diluted 1/1000 in the B-Per after the
Mg and Ca to 10 units per mL final). DNase I requires calcium in addition to Mg++ and works better in low salt
conditions (frequently overlooked facts, Ambion's web site has some good info).
See: Sauer:Lysing E. coli with Lysozymes (http://openwetware.org/wiki/Sauer:Lysing_E._coli_with_Lysozymes)
So, You have a mL or so of cells in a microfuge tube, harvest them in the cold and place on ice, and then aspirate the
medium (aspiration works better than pipette and is faster). Go just above the pellet, but not TO the pellet. The liquid
will rise up the wall the of the tube and get sucked away leaving a practically dry pellet.
Pipette a fixed defined volume of the lysis solution to each sample (generally 50-100 ?L) and vortex into solution at
room temp. Place the tube in a rack at room temp and proceed to the next pellet. After resuspension, the cells will
clear in less than a minute when placed at room temp.
So now you have intact ribosomes in a low-salt solution. Add about 200-400 uL of salty extraction buffer. This solution
chelates all of the Mg++, disrupts ribosomes, increases ionic strength, and provides a carrier for precipitation. I use 50
mM bis-Tris pH 6.5, containing 400 mM NaCl, 5 mM EDTA and 1 ?L/400 ?L of linear polyacrylamide (LPA)at 10
mg/mL.

Organic Extraction and Precipitation

Add about 300-500 ?L of acidic phenol/chloroform (I buy Ambion's, I still have the same bottle I bought years ago). If
you're adding a control RNA for quantification, now is when you do it. Vortex the samples, let them stand about 5 min
on the bench. Spin a few minutes and remove the supernatant to a new tube. If I need cleaner RNA, I repeat the
extraction, if I'm just running denaturing gels, I precipitate.
Add slightly-greater-than-equal volume of isopropanol to each sup., vortex. I see different opinion about the temp this
should be done. I put my tubes on ice for 30 minutes or overnight at -20. Then I spin for 30-45 minutes. I think it was
Ambion's site where they showed "spin time" as the most critical factor. Either your precipitation conditions cause large
aggregates that pellet fast, or small aggregates than pellet slow, a long spin here covers both.
Aspirate the isopropanol (don't go near the pellet, it'll jump into your tip and go bye-bye). Wash with ~700 ?L of 75%
Ethanol to de-salt, re-spin, aspirate, then wash with 95% ethanol to dehydrate, re-spin, aspirate, and place the tubes
on their side on the bench to dry. Before resuspending the pellets MAKE SURE they are dry. It seems a small amount
of ethanol will cause distortion in the gels, probably from partial precipitation. Be careful, when they are dry, static can
cause the RNA pellet to fly out, just keep your eyes on the prize.
After drying, resuspend either in non-denaturing solution (10 mM bis-Tris, pH 6.5, 0.1 mM EDTA) or denaturing
solution (95% stabilized formamide, 10 mM bis-Tris pH 6.5, 0.1 mM EDTA, some bromophenol blue to taste). About
50 ?L for each mL of culture harvested. Expect 50-100 ?g of total RNA per 10^9 cells. I run about 1 ug total or 0.5 ?g
ribosomal RNA per well when staining with Sybr-Green II.
For determining the concentration of your RNA, see this article:Sauer:Nucleic acid quantitation
(http://openwetware.org/wiki/Sauer:Nucleic_acid_quantitation)

http://dualsystems.freshdesk.com/support/articles/8238-isolation-of-total-rna-from-escherichia

15/10/2014

Isolation of total RNA from Escherichia coli :

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http://dualsystems.freshdesk.com/support/articles/8238-isolation-of-total-rna-from-escherichia

15/10/2014