Molecular Biology Techniques Q&A

This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “RNA Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.

tRNA Isolation

What is the best way to isolate tRNA from bacteria? (Thread 32357)

Q I need to isolate E. coli tRNA for use in a cell-free protein synthesis system. But first, total RNA needs to be isolated. I found two suitable procedures for isolating total RNA: one uses acid guanidinium thiocyanate and phenol/chloroform extraction (Chomczynski & Sacchi) and the other uses only Tris-HCl-saturated phenol extraction (Cayana et al.). I know guanidinium thiocyanate inhibits DNase and RNase, but are there additional advantages compared to the Tris-phenol method? Which procedure would you recommended in my case?

A For total RNA extraction, the guanidinium thiocyanate method is simpler and more widely used. You could use commercial Tri-Reagent or Trizol, which contains a dye to help with phase separation. For separating tRNA, if you are not using column purification, the guanidinium thiocyanate method will be better since it removes most contaminating DNA.

Q Why does the Chomczynski & Sacchi method use guanidine thiocyanate, phenol, and chloroform, whereas the alternate method from Cayana et al. uses only phenol?

I searched the literature and had difficulty finding proper information about this topic. This is partly because during the time the original protocols for nucleic acid preparation and isolation were being developed, authors referred to “soluble RNA” or “sRNA,” not “tRNA.” Most papers on RNA extraction described procedures for mammalian cells, usually for mRNA extraction for transcriptional analysis. These procedures can't be applied easily to E. coli tRNA for use with coupled in vitro transcription/translation.

It seems that there are three main categories for nucleic acid isolation: total DNA and RNA isolation; total RNA without DNA (including insoluble, large RNA molecules and soluble, small ones such as rRNA and mRNA); and soluble, small RNA molecules without large ones (including tRNA, aminoacylated tRNAs, and unmodified tRNA).

Soluble, small-size RNA molecules such as tRNA can be extracted directly from bacterial cells without cell lysis using phenol. This approach depends on phenol permeabilization of cell membranes, making it possible for small particles like tRNA to pass through, while excluding mRNA, rRNA, and other larger molecules. To isolate total RNA, including rRNA or mRNA, acetone should be added to the phenol extraction procedure to increase membrane permeability to a point where larger RNAs can diffuse out of the cell. Using phenol alone should be limited to bacteria (or possibly yeast) cells.

For extraction of nucleic acids from mammalian cells, the development of protocols using guanidine thiocyanate/phenol/chloroform to lyse the cells and deactivate nucleases and antioxidants was necessary.

A Phenol was originally used for nucleic acid extraction because it denatures proteins, including nucleases. Use of guanidium thiocyanate (a more powerful denaturant than phenol) was developed later for use with tissues possessing high levels of RNase, such as pancreas. Guanidium thiocyanate later became a popular reagent for RNA extraction from most tissues. Of course, if you are only interested in small RNAs, you should use a method developed specifically for that purpose.

Q I am also trying to isolate tRNA for use in a cell-free protein expression system and haven't been able to find answers in the literature. Did you try using only phenol to extract tRNA from E. coli? Did it work, and if so, would you mind sharing your protocol? Also, how did you check the quality of your tRNA preparation?

A Yes, phenol extraction worked very well. Among the procedures commonly employed to purify tRNA, including stepwise precipitation, sucrose gradient centrifugation, gel filtration, and column chromatography on DEAE-cellulose, the precipitation method is definitely the most convenient. To assess the resolution profile, I treated aqueous solutions of phenol-extracted crude tRNA stepwise with IPA and collected the precipitates by centrifugation after each step. Agarose gel electrophoresis and ethidium bromide visualization were used to detect the RNA species contained in the pellet.

I extracted RNA from E. coli K12, E. coli BL21(DE3), and Cupriavidus necator (Ralstonia eutrophus) H16 cells using the Cayana et al. procedure. I equilibrated the phenol with Buffer A: 1 mM Tris-HCl, pH 7.2, 10 mM Mg(OAc)₂. After the cells (∼5 mL) were thawed in 1 volume of Buffer A, I added 1.5 volumes of equilibrated phenol and extracted by repeated vortexing over a period of 30 min with constant cooling on ice. I separated the phases by centrifugation (8600× g, 10 min) and removed the aqueous layer carefully with a pipette. After a second extraction, I combined the aqueous layers in a new tube and ethanol precipitated the RNA. I resuspended the pellet in 5 mL 0.05 M NaCl and stored the tubes at -20°C.

The next day, I added LiCl to a concentration of 0.8 M and collected the precipitate by centrifugation. I moved the supernatant to another tube and adjusted the pH. Addition of IPA led to precipitation of tRNA, and the resulting pellet was washed with 96% EtOH and resuspended in 0.05 M NaCl. I repeated this 4 more times, adding 1, 1, 5, and 15 mL IPA, respectively.

tRNA was visible on a gel, as well as the rRNA subunits and a >100,000 bp contaminant, which were removed after the third IPA precipitation step.

I checked the quality and quantity of the recovered fractions by spectrophotometry, using the 260/280 nm and 260/230 nm ratios.

I have repeated this procedure 3 times now, and each time I obtained 2-3 mL of the tRNA fraction from 500 mL OD 3-4 culture in 2× YTPG medium. Each time, the tRNA concentration was ∼13 mg/mL with values of 2.0 +/- 0.1 for both ratios. This is quite good and in all cases was suitable for in vitro experiments.

In addition to what I described, I am planning to use 2-D denaturing urea PAGE and ultrasensitive silver staining to assess my tRNA fractions.

If you try this method, keep in mind that you need to resuspend the tRNA in pure ddH₂O. Do not use TE buffer as advised by most protocols because EDTA will dissociate your ribosomes into their subunits.