Technical Bulletin: #160
|The Use of LiCl Precipitation for RNA Purification |
LiCl has been frequently used to precipitate RNA, although precipitation with alcohol and a monovalent cation such as sodium or ammonium ion is much more widely used. LiCl precipitation offers major advantages over other RNA precipitation methods in that it does not efficiently precipitate DNA, protein or carbohydrate (Barlow et al., 1963). It is the method of choice for removing inhibitors of translation or cDNA synthesis from RNA preparations (Cathala et al., 1983). It also provides a simple rapid method for recovering RNA from in vitro transcription reactions.
Ambion provides LiCl as an RNA recovery agent in its MEGAscript® and mMESSAGE mMACHINE® large scale in vitro transcription kits. However, while providing telephone technical service, we have noticed that many users are reluctant to use LiCl, presumably because there is not good data in the literature describing its properties. We have conducted a systematic study of the use of LiCl and find that it is a very effective method for precipitating RNA, especially from in vitro transcription reactions.
The three key variables we studied were: (a) the temperature at which the precipitate is allowed to form, (b) the concentration of the RNA and the lithium chloride used and, (c) the time and speed of centrifugation used to collect the precipitated RNA. All of these variables have been explored and are discussed below. We find that LiCl precipitated RNA samples prepared in this way require no further purification for use in hybridization and in vitro translation reactions. It has been reported that lithium chloride is unsuitable for cell free translations due to the inhibition of chloride ions (Maniatis, et al., 1989); However, we have not been able to document any deleterious effect in either translation or microinjection experiments. Another advantage is that lithium precipitation efficiently removes unincorporated NTPs, which allows for more accurate quantitation by UV spectrophotometry.
Comparison of Lithium Chloride and Ammonium Acetate/Ethanol
Precipitation Parameters of Lithium Chloride RNA Concentration
Lithium Chloride Concentration
Figure 2. Effect of Lithium Chloride Concentration on Precipitating RNA. Lane 1, RNA size standards. Lane 2, 2.5 M LiCl. Lane 3, 1.0 M LiCl. Lane 4, 0.5 M LiCl, and lane 5, no LiCl.
Figure 3. Effect of Precipitation Temperature Using Lithium Chloride. Lane 1, RNA size standards. Lane 2, RNA centrifutged immediately without chilling. Lane 3, RNA chilled at -20°C for 30 minutes before centrifugation. Lane 4, RNA incubated at 25°C for 30 minutes to test precipitation time independently of chilling. Lane 5, RNA chilled at -20°C for 1 hour. Lane 6, RNA incubated at 25°C for 1 hour.
Figure 4. Effects of Centrifugation Time in Precipitating RNA. Lane 1, RNA size standards. Lane 2, RNA centrifuged for 20 minutes, Lane 3, 10 minutes, Lane 4, 5 minutes, Lane 5, 2 minutes, Lane 6, 1 minutes, and Lane 7, 30 seconds.
Contrary to previously published reports, we find that lithium chloride does not appear to preferentially precipitate higher molecular weight RNA rather than smaller RNA. Lithium chloride precipitations using mixtures of equal amounts of RNA of lengths 100, 200, 300, 400, and 500 bases (RNA Century™ Size Standards) showed that all sizes were precipitated equally well (data not shown). Since it was thought that the larger sizes might aid in the precipitation of smaller size transcripts, the experiments in this paper were performed using each size of transcript separately. No differences in precipitating a single size of RNA (e.g. 100 bases) as compared to a mix of all sizes of the RNA markers was seen. It should be noted, however, that some small RNAs such as tRNAs are not efficiently precipitated by lithium chloride. This is likely due to the high degree of secondary structure in tRNA. While we recommend the routine use of lithium chloride for precipitating RNA from solutions containing at least 400 µg/ml RNA, we are cautious about recommending its use with lower concentrations of RNA until we have tested its use with a wider range of RNAs.
- Barlow, J.J., Mathias, A.P., Williamson, R., and Gammack, D.B., (1963). A Simple Method for the Quantitative Isolation of Undegraded High Molecular Weight Ribonucleic Acid. Biochem. Biophys. Res. Commun. 13:61-66.
- Cathala, G., Savouret, J., Mendez, B., West, B.L., Karin, M., Martial, J.A., and Baxter, J.D., (1983). A Method for Isolation of Intact, Translationally Active Ribonucleic Acid. DNA 2:329-335.
- Maniatis, Sambrook, Fritsch, (1989). Molecular Cloning: A Laboratory Manual 2nd ed., Vol. 3, Appendix E.12.