RNA Basics: Working with RNA

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.

• Clean gloves should always be worn when handling any reagents, reaction vessels, or samples. Gloves should be changed frequently, especially after touching refrigerator handles, doorknobs, or any lab equipment such as pipettes.
• When performing procedures that use RNase (e.g., ribonuclease protection assays and plasmid purification), care should be taken that pipettors are not contaminated by accident. One potential source of contamination is the metal tip ejector mechanism on the side of the pipettor. Removing the metal ejector bar and wiping the pipettor, pipettor bar, and ejector with an RNase decontamination solution between experiments will help eliminate this concern.
• Autoclave tips, tubes, and solutions to help inactivate RNases. Glassware can be baked at 300°C for four hours and plasticware, tubes, and most solutions can be DEPC-treated.

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.

Learn more about RNase-free 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).

Learn more about RNase-free buffers and reagents
Learn more about nuclease-free water

Supplement the above methods by employing the use of RNase decontamination reagents. They can be used on lab glassware, plastic surfaces, countertops, and pipettors. Reagents can be poured onto, sprayed, or wiped over surfaces and work immediately upon contact. Treated labware is simply rinsed twice with distilled water and is ready for use.

Learn more about RNase decontamination

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.

Learn more about RNase detection
Learn about RNase control

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.

• Add DEPC to solutions at a concentration of 0.05–0.1% (e.g., add 0.5–1 mL DEPC per liter)
• Stir or shake DEPC into solution and incubate for several hours
• Autoclave for at least 45 minutes
• Compounds containing primary amine groups, such as Tris (2-Amino-2-hydroxymethyl-1,3-propanediol), will react with DEPC, and thus should be added only after DEPC treatment is complete

Learn more about RNase and DEPC

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⁺) [1]. 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.

Learn more about the basics of RNA isolation
Read tips about handling RNA
View ten ways to improve your RNA isolation

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.

Read more about quantitating RNA
Learn more methods to check RNA integrity

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.

Table 1. Ribosomal subunit sizes in representative eukaryotes

Read more about RNA ladders and markers
Tips for visualizing RNA markers

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.

For short-term RNA storage:

• RNase-free H₂O (with 0.1 mM EDTA)
• TE buffer (10 mM Tris, 1mM EDTA)

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.

Learn more about RNA stabilizers

1. Wallace, DM. (1987) Precipitation of Nucleic Acids. Methods of Enzymology 152:41–46. 
2. Chomczynski, P. (1992) Solubilization in formamide protects RNA from degradation. Nucleic Acids Research 20:3791–3792.