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Working with RNA

An RNase free environment is essential when working with RNA samples. In the laboratory, obtaining full length, high quality RNA can be challenging. There are two main reasons for RNA degradation during RNA analysis. First, RNA by its very structure is inherently weaker than DNA. RNA is made up of ribose units, which have a highly reactive hydroxyl group on C2 that takes part in RNA-mediated enzymatic events. This makes RNA more chemically labile than DNA. RNA is also more prone to heat degradation than DNA. Secondly, enzymes that degrade RNA, ribonucleases (RNases) are so ubiquitous and hardy; removing them often proves to be nearly impossible. For example, autoclaving a solution containing bacteria will destroy the bacterial cells but not the RNases released from the cells. Furthermore, even trace amounts of RNases are able to degrade RNA. Therefore, it is essential to avoid inadvertently introducing RNases into the RNA sample during or after the isolation procedure.

Sources of RNases

Skin: The presence of RNases on human skin surfaces has been well documented. RNase contamination through this source is very easy to acquire and spread if tubes, pipette tips, bench tops, etc. are touched with bare hands.

Dust: Dust particles floating in the air often harbor bacteria or mold. The RNases from these microorganisms are deposited wherever the dust settles. This includes lab equipment, open bottles, etc.

Reagents: If the reagents used for RNA analysis are not certified to be RNase free, there is a good chance that some of the contamination will come from this source. Reagents can also become contaminated in the lab itself if proper care is not taken.

Samples: RNase contamination can come from the samples themselves as tissues and cells contain endogenous RNases.

How to maintain an RNase-free environment

Gloves: Always wear sterile disposable gloves when handling reagents and RNA samples. It is important to remember that once the gloves have touched equipment in the lab such as centrifuges, pipettes and door handles, they are no longer RNase free. Change gloves frequently as the protocol progresses from crude extract to more purified material.

Disposable sterile plasticware: Disposable plasticware greatly reduces the possibility of contaminating your samples and are typically RNase-free (check with supplier). In the event of a contamination, it also minimizes spread of the contamination. The use of disposable tips, tubes, etc. is therefore highly recommended.

Non-disposable plasticware: Non-disposable plasticware should be treated before use to ensure that it is RNase-free. Plasticware should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA followed by RNase-free water. Alternatively, chloroform-resistant plasticware can be rinsed with chloroform to inactivate RNases.

Good quality reagents: Always ensure that all reagents and chemicals purchased commercially are guaranteed to be RNase free. Testing each batch before use may be a prudent step.

DEPC-treated water: Use DEPC-treated water instead of regular PCR grade water. DEPC inactivates RNase by histidine modification of the bases. If DEPC-treated water is made in-house, always remember to autoclave before use to degrade the DEPC.

RNase inhibitors: The use of RNase inhibitors is highly recommended with samples containing endogenous RNase. Use RiboSafe RNase Inhibitor (BIO-65027), which completely inhibits a broad spectrum of eukaryotic RNases, including RNases A, B and C.

Aseptic techniques: Always use proper microbiological aseptic techniques when working with RNA.

Decontamination techniques: Heat-proof glassware should be cleaned with a detergent and rinsed thoroughly prior to being baked at 180°C for several hours to inactive RNases. Autoclaving alone will not inactivate many RNases. Polycarbonate or polystyrene materials can be decontaminated by soaking in 3% hydrogen peroxide for 15 minutes, followed by thorough rinsing with RNase-free water.

Storage: Correct storage of RNA is also very important to avoid RNA degradation. In the short-term, RNA may be stored in RNase-free water or TE buffer at -80ºC for 1 year without degradation. For long-term storage RNA samples may be stored as ethanol precipitates at -20°C. However, when dissolved in ethanol, RNA is not dispersed evenly in the solution and cannot be used directly in quantitative experiments. Instead, precipitates should be pelleted and redissolved in an aqueous buffer before pipetting. When working with RNA on the bench, keep the RNA aliquot on ice and the lid closed when performing other steps.

Determination of RNA yield, purity and integrity

The yield of total RNA may be determined spectrophotometrically at 260nm, whereby 1 unit of absorbance (A260) = 40µg of single stranded RNA/ml. The purity can also be determined spectrophotometrically from the ratio of the relative absorbance at 260 and 280nm. Good quality RNA will have an A260/A280 ratio in the range of 1.7 to 2.1.

RNA integrity can be assessed by running 2-4µg of a total RNA sample on an agarose denaturing gel. The RNA may be visualized by ethidium bromide staining, which reveals the ribosomal RNA bands. These bands can vary depending on the organism the RNA was extracted from. In general, for good quality RNA the bands should be distinct, with no smearing underneath them and the 28S band (larger) should be approximately twice as intense as the 18S band.

Alternatively, RNA quality and integrity can be checked using commercially available automated systems: the 2100 Bioanalyser (Agilent) or Experion (Bio-Rad). Both platforms determine RNA quality using a numerical system which represents the integrity of RNA. The Bioanalyzer offers the RIN algorithm (RNA Integrity Number) on the Bioanalyzer 2100 whereas the Experion offers an algorithm for calculating the RNA Quality Index (RQI).