Isolating RNA is easy and fun.

Isolating RNA is easy and fun. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol33/iss1/2 Akins, R.A., A.R. Kubelik and We reported previously the development of a general method for cloning Neurospora nuclear genes A.M. Lambowitz by sib selection, using a library of N. crassa genomic DNA fragments in plasmid pRAL1 (Akins Map of Plasmid pRAL1 and Lambowitz Mol. Cell. Biol. 5:2272-2278, 1985). Fig. 1 is a revised map of plasmid pRAL1. The size of the plasmid is measured more accurately as 4.7 kb, rather than 4.4 kb reported previously. In addition, the position of the EcoR1 site in the qa-2^+ gene was indicated incorrectly in the previous map. The sizes of the EcoR1 fragments are 2.8 and 1.85 kb. We and others have now cloned at least ten genes using the pRAL1 library: nic-1 and inl (Akins and Lambowitz, Molec. Cell. Biol. 5:2272-2278, 1985), cyt-18 (Akins and Lambowitz, unpubl.), cyt-(297-24) Kuiper, de Vries, Akins and Lambowitz, unpubl.), cyt-4 (Serizawa, Akins and Lambowitz, unpubl.), cyt-(289-4) (Kubelik and Lambowitz, unpubl.), his-2 (Akins, Lambowitz and Kinsey, unpubl.), van (Mann, Metzenberg, Akins and Lambowitz, unpubl.), cys-3 (Paietta, Marzluf, Akins and Lambowitz, unpubl.) and met-7 (Dr. M. Case, University of Georgia, personal communication). The library is available to all investigators. This work supported by NIH grant GM23961. Dept. of Biochemistry, St. Louis University School of Medicine, St. Louis, MO 63104 Chambers, J.A.A. and V.E.A. Russo We have found that the common phenol or guanidium thiocyanate based procedures for isoIsolating RNA is easy and fun lating RNA simply do not repay the effort for the yields involved (0.2-0.3 mg total RNA per g fresh weight with guanidium salts). A number of observations led us to examine and slightly modify a procedure originally developed for tissue culture cells (Auffray and Rougeon, 1979, Eur. J. Biochem. 107:303). This somewhat unusual procedure is based upon lithium chloride/urea solubilization of the cellular contents and the ultimate precipitation of RNA at high ionic strength. The combination of effective solubilization, inhibition of RNAse activity, effective precipitation of RNA, and a minimum of handling results in higher yields (0.5-1 mg RNA per g fresh wt.) for much less effort.

Isolating RNA is easy and fun. Isolating RNA is easy and fun.

Abstract Abstract
Isolating RNA is easy and fun.
This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol33/iss1/2 Chambers, J.A.A. and V.E.A. Russo We have found that the common phenol or guanidium thiocyanate based procedures for iso-Isolating RNA is easy and fun lating RNA simply do not repay the effort for the yields involved (0.2-0.3 mg total RNA per g fresh weight with guanidium salts). A number of observations led us to examine and slightly modify a procedure originally developed for tissue culture cells (Auffray and Rougeon, 1979, Eur. J. Biochem. 107:303). This somewhat unusual procedure is based upon lithium chloride/urea solubilization of the cellular contents and the ultimate precipitation of RNA at high ionic strength. The combination of effective solubilization, inhibition of RNAse activity, effective precipitation of RNA, and a minimum of handling results in higher yields (0.5-1 mg RNA per g fresh wt.) for much less effort.

Precautions
General precautions for handling RNA are applied.
With the exception of the lithium chloride/urea solution -which is prepared fresh -all aqueous solutions are autoclaved before use. We are not enthusiastic about the use of dietheyl pyrocarbonate (DEP) as a cure-all for RNAse problems and in any case it is not compatible with Tris buffers. The lithium chloride/urea solution can be neither autoclaved nor treated with DEP.
The combination of chaotropism and high ionic strength seems to be completely effective in inhibiting RNAses.
2. Pulverize the frozen mycelium under liquid nitrogen in a mortar and pestle.
3. Resuspend the mycelium in 7-10 volumes of 6M urea, 3M LiCl. This may freeze, but this is unimportant, because it will thaw fairly quickly.

4.
Pour the suspension, which should be fairly viscous, into the homogenizer vessel (50 ml vessel of the Sorvall omni-mixer) and wash the mortar out with an equal volume of urea/LiCl. 5. Homogenize the mycelium. In the 50 ml vessel of the Sorvall Omn-Mixer, we use 2 x 30-60 s on ice to homogenize effectively. Any conditions that produce a useful homogenate for other extractions should do.
6. Centrifuge the homogenate. We use 10,000 rpm (16,000 g max) in the Sorvall HB4 rotor for 20 min, 4° C. It is better to use a swinging bucket rotor because the resulting pellet is more compact and firmer. A clean separation between pellet end supernatant is very important. Ignore any lipid layer on the supernatant. 7. Decant the supernatant and store at -20° C overnight. This may freeze, but it does not seem to affect yields.
8. Centrifuge as before. Discard the supernatant and drain the pellet thoroughly.
9. Resuspend the pellet by vigorous vortexing with 5 ml 0.5% SDS, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. The suspension is often milky and viscous. If the pellet does not break up effectively, the suspension can be centrifuged (10,000 rpm, 16,000 g max 10 min 4° C, Sorvall HB4 rotor) and the pellet resuspended and extracted. This appears to give some improvement in the A260/A230 ratio (a measure of carbohydrate contamination).
10. Extract the suspension or pooled supernatants once with an equal volume of chloroform/isoamyl alcohol. It is important to obtain a good emulsion. Vortex vigorously 4 to 5 times for 15-30 s. Centrifuge (5000 rpm, 4000 g max, 10 min 4° C, Sorva11 HB4 rotor) to separate the phases.
11. Remove the aqueous phase, add ammonium acetate to 2.5 M and two volumes of ethanol to the aqueous phase. Precipitate the nucleic acids for 1 h at -70° C. Recover the precipitate by centrifugation (10,000 rpm, 20 min, 4° C, Sorvall HB4 rotor), wash the pellet once or twice with 70% ethanol and dry under vacuum.
12. Redissolve the pellet in the appropriate buffer for your next step.

Oligo (dT) cellulose chromatography
We have used protocols described by Schleif and Wensink (Practical Methods in Molecular Biology, Springer Verlag, 1981, pp. 168-170) and by Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982, pp. 197-198) to purify polyA^+ mRNA and find that they work equally well. It is important not to load more than 10 mg of total RNA per gram of oligo (dT) cellulose. If you find a poor yield of poly A^+ RNA (less than 1% of total RNA) it is possible that the column was overloaded. In that case we find that the non-binding (poly A-) fraction can be reapplied to a column after washing the column with alkali and buffer as described in the references cited above and more poly A+ RNA recovered from it. As judged by stimulation of in vitro protein synthesis and the pattern of translation products this second (and even third) extraction is as good as that obtained on the first pass.
With reasonable yields of poly A^+ RNA (..20 ug at 4-5 ug ml^-1) we do not need to add carrier for ethanol precipitation. With smaller quantities we use Sigma tRNA, type V, and about 100 ug ml^-1 as carrier.
In general, we find that the RNA from step 12 has an A260/A280 of 2-2.1 and A26O/A230 of 2-2.2. The poly A+ content is about 1.5% of total. There is very little DNA as judged by agarose gel-electrophoresis. As judged by methyl mercury agarose gel electrophoresis, the RNA is up to 4000 nucleotides long with little evidence of degradation of the ribosomal RNAs.
Using rabbit reticulocyte lysate supplied by Amersham for in vitro translation (Cat. No. N90, 0.2 ug poly A^+ in 2 ul double distilled H20 + 10 ul reticulocytes) we typically get a 40-fold stimulation of incorporation with polypeptides in excess of 200 kd being made. Depending upon the protocol used, we find that cDNA with median lengths between 700 and 1300 nucleotides is made.
A rider to be added is that we are isolating RNA from cultures growing under rather benign conditions. If cultures are grown under conditions likely to induce high levels of nucleases (e.g. phosphate or nitrogen limitation) then there may be problems with nucleases. In that case we stress the rapid freezing in liquid nitrogen and suggest homogenising the mycelium in a larger volume of urea/lithium chloride (say 25-40 vols) to dilute the nucleases.