Researchers who work with RNA know that RNA is a sensitive molecule that gets rapidly degraded. Because of the single-stranded nature of RNA, it is easily accessible to proteins to inactivate and target for degradation. Ribonucleases (RNases) are ubiquitous enzymes that target RNA for degradation into smaller components. Important considerations when working with RNA include ways in preventing RNase contamination, how to properly precipitate and store RNA, and how to determine RNA yield.
There are several ways to prevent RNase contamination in the laboratory. Sterile technique goes a long way: use clean gloves and a clean lab coat, keep tubes and bottles closed, use a clean (RNase-free) work area, and ensure you use RNase-free supplies, solutions, and reagents.
To identify if RNase contamination has occurred in your lab, RNase detection kits are available. These kits help allow researchers to identify contaminated reagents and equipment quickly.
To ensure that solutions are free of RNase contamination, they can be treated with diethylpyrocarbonate (DEPC). DEPC reacts with histidine residues of proteins and will inactivate RNases. However, it can also react with RNA, so it needs to be removed by heat treatment before the solution is used.
WARNING: DEPC is a suspected carcinogen so appropriate precautions should be taken when handling: always wear gloves and handle under an approved fume hood.
Common lab supplies are often overlooked as a source of RNase contamination. This is why it is important to use RNase-free lab supplies, such as pipette tips and tubes.
Similar to lab supplies, common lab buffers, reagents, and water are overlooked as sources of RNase contamination. Many buffers and reagents are offered in an RNase-free format. Water is also offered as nuclease-free (both RNase- and DNase-free).
There are two main methods of precipitating RNA. The most common way researchers precipitate and purify RNA is via alcohol precipitation. Another way of precipitating RNA is with the use of lithium chloride.
Precipitating RNA with alcohol (ethanol or isopropanol) requires a minimum concentration of monovalent cations (e.g., 0.2 M Na+, K+; 0.5 M NH4+) . After the salt concentration has been adjusted, RNA may be precipitated by adding 2.5 volumes of ethanol or 1 volume of isopropanol and mixing thoroughly, followed by chilling for at least 15 minutes at –20° C.
The role of isopropanol in RNA isolation is to precipitate RNA. RNA is insoluble in alcohols such as isopropanol and ethanol. While isopropanol is somewhat less efficient at precipitating RNA than ethanol, isopropanol in the presence of NH4+ is better than ethanol at keeping free nucleotides in solution, and so separating them from precipitated RNA. RNA precipitation is faster and more complete at higher RNA concentrations. RNA concentrations of 10 µg/ml can usually be precipitated in several hours to overnight with no difficulty, but at lower concentrations a carrier nucleic acid or glycogen should be added to facilitate precipitation and maximize recovery.
Lithium chloride (LiCl) is an alternative method of precipitating RNA, and has the advantage of not precipitating carbohydrates, proteins, or DNA. LiCl is frequently used to remove inhibitors of translation which co-purify with RNA prepared by other methods. A final LiCl concentration of 2–3 M is needed to precipitate RNA (adding an equal volume of 4 M LiCl, 20 mM Tris-HCl, pH 7.4, and 10 mM EDTA). Note that no alcohol is needed for LiCl precipitation of RNA. RNA should be allowed to precipitate at –20°C; precipitation time depends on RNA concentration. It is generally safe to allow the RNA to precipitate for several hours to overnight.
After centrifugation to collect the RNA, pellets can be rinsed with 70% ethanol to remove traces of LiCl. LiCl efficiently precipitates RNA greater than 300 nucleotides in length. While LiCl can effectively precipitate RNA from more dilute solutions, it’s a good rule of thumb that the RNA concentration should exceed 200 µg/mL.
Total RNA yield can be measured with a spectrophotometer at the absorbance of 260 nm where one unit of absorbance is (A260) is 40 µg RNA/mL.
RNA size markers are used to visualize RNA. They can be unlabeled for staining with EtBr or biotinylated for subsequent secondary detection.
Known RNA transcripts and double-stranded DNA markers can also be end-labeled with polynucleotide kinase (5’ end-labeling reaction) or Klenow fragment (3’ filling reaction) and denatured for use as labeled size markers.
Other guides to RNA size and migration position are the xylene cyanol and bromophenol blue dyes present in most loading buffers, and rRNA species present during electrophoresis of total RNA for northern analysis. The migration position of the dyes included in loading buffers is affected both by gel percentage and composition (denaturing vs. nondenaturing). Ribosomal RNA comprises 80% of total RNA samples. Both the 18S and 28S species are strongly visible in northern gels stained with EtBr or UV-shadowed. Table 1 gives their sizes in several different vertebrate species.
|Average number of bases|
RNA may be stored in a number of ways, depending on if it is needed for short-term or long-term storage. RNA is generally stable at –80° C for up to a year without degradation. Magnesium and other metals catalyze non-specific cleavages in RNA, and so should be chelated by the addition of EDTA if RNA is to be stored and retrieved intact.
NOTE: It is important to use an EDTA solution known to be RNase-free for this purpose. Older EDTA solutions may have microbial growth which could contaminate the RNA sample with nucleases. It has been suggested that RNA solubilized in formamide may be stored at –20°C without degradation for at least one year .
For short-term RNA storage:
For long-term RNA storage, the use of RNA stabilization reagents is recommended. RNA stabilizers help preserve the integrity of RNA during RNA sample collection or post-collection.
For Research Use Only. Not for use in diagnostic procedures.