All reagents and equipment must be specially treated to inactivate Cases prior to use. Even if all the reagents have been decontaminated, Cases can be reintroduced by contact with unloved hands or with unfiltered air.
Electrophoresis tanks for RNA analysis can be cleaned by wiping them with a solution of SDS (1%), rinsing with water, then rinsing with absolute ethanol, and finally soaking them in 3% H2O2 for 10 minutes. Rinse tanks with DEC (dimethyl polycarbonate)-treated and autoclave water before use (see below).
Soak plastic ware (2 hours, +37 °C) in 0.1 M NaOH/1 mm EDTA (or absolute ethanol with 1% SDS), rinsed with DEC or DMPC (dimethyl polycarbonate) treated water and heated to +100 °C for 15 minutes in an autoclave. Workspace and working surfaces Designate a special area for RNA work only.
Treat surfaces of benches and glassware with commercially available RNAS inactivating agents. Also, wipe benches with 100% ethanol each time prior to use, in order to rid the area of microorganisms.
All solutions, except This buffers, should be treated with 0.1% DEC (or DMPC) overnight at room temperature and then autoclave. Protector RNAS Inhibitor is fully active over a broad temperature range of +25 to +55 °C.
To keep the inhibitor active, avoid temperatures above +60 °C, solutions containing strong denaturing agents (such as SDS or urea), and maintain reducing conditions (1 mm DTT). To protect difficult RNA samples, the amount of Protector RNAS Inhibitor can be increased up to 16 times the standard concentration without interfering with the performance of enzymes used in the assay.
For the reasons mentioned above, RNA is very susceptible to degradation when left at room temperature. Dissolve RNA by adding RNase-free buffer or water, then standing the tube on ice for 15 minutes.
Instead, to melt out secondary structures, heat RNA to +65 °C for 15 minutes in the presence of denaturing buffers. You will receive the Roche Newsletter for our new and exciting products and special offers.
RNA is more susceptible to degradation than DNA, due to the ability of the 2´ hydroxyl groups to act as nucleophilic. Many ribonucleases (Cases) bypass the need for metal ions by taking advantage of the 2´ hydroxyl group as a reactive species.
Aqueous solutions, reagents used in experiments Environmental exposure, Cases are in the air, most surfaces and dust Human contact with hands and skin New England Bio labs' enzymes certified for RNA work have been purified free of ribonucleases.
RNAS contamination can be prevented by following a few common sense laboratory procedures: Always wear gloves during an experiment and change them often, especially after contact with skin, hair or other potentially RNase-contaminated surfaces such as doorknobs, keyboards and animals.
Autoplaying will destroy any unreacted DEC which can otherwise react with other proteins and RNA. Decontaminate polycarbonate or polystyrene materials (e.g. electrophoresis tanks) by soaking in 3% hydrogen peroxide for 10 minutes.
As an alternative to the historic use of DEC, which can inhibit enzymatic reactions if not completely removed, we have found that Million (Millipede) purified water is sufficiently free of Cases for most RNA work. [Note: Compounds with primary amine groups (e.g., This) which will react with DEC, cannot be DEPC-treated.
Solutions and buffers (e.g. DTT, nucleotides, manganese salts) should be prepared by dissolving the solid (the highest available purity) in autoclave DEPC-treated or Million water and passing the solution through a 0.22 µm filter to sterilize. It has the same inhibition profile of human or porcine inhibitors but is more stable due to improved resistance to oxidation (4).
The inhibitor requires low concentration of DTT (< 1 mm) to maintain activity, making it ideal for reactions where low DTT concentration is required (e.g., real-time RT-PCR). Ribonucleoside Vandal Complex (NEB #S1402) is a transition-state analog inhibitor of RNAS A-type enzymes with K i = 1 × 10 -5 M. This complex is compatible with many RNA isolation procedures, but it should not be used in the presence of EDTA.
To save your cart and view previous orders, sign in to your NEB account. Cases, which play important roles in nucleic acid metabolism, are found in both prokaryotes and eukaryotes, and in practically every cell type.
The human body uses Cases to defend against invading microorganisms by secreting these enzymes in fluids such as tears, saliva, mucus, and perspiration. Thus, the use of unloved hands could easily result in RNAS contamination compromising critical experiments.
Always wear a laboratory coat to prevent particulate materials from falling from your clothing onto your sample. Avoid working with RNA where airflow is turbulent or near surfaces that can create particulates (like a chalkboard).
However, tips and tubes can be an easily overlooked source of potential RNAS contamination. Each lot of tips and tubes is rigorously tested for RNAS and Name contamination and is certified nuclease-free.
When glassware and metalware are required, treat them with Nitrogen RNAS Zap ™ reagent or wipes. The baking procedure typically involves incubation in an oven at 450 °F for 2 hours or more (don't include tape, as it will burn!).
When 2 hours is too long to wait, treating with RNAS Zap reagent or wipes is a great alternative. Just make sure that the RNAS Zap reagent is not in contact with surfaces of forceps, spatulas, or other reactive metalware (e.g., aluminum) for more than a few minutes, as this could lead to corrosion.
Mark baked and RNAS Zap reagent–treated items as “RNase-free” to distinguish them from untreated pieces. We recommend storing the treated equipment in a clearly marked RNase-free zone to prevent accidental contamination.
Due to the ubiquitous nature of Cases, depending on the source and upkeep of equipment used to provide the water, the water and buffers used in molecular biology applications can be frequent sources of RNAS contamination. Diethylpyrocarbonate (DEC) treatment is the most common method used to inactivate Cases in water and buffers.
We provide a variety of Nitrogen buffers and water (DEPC-treated or untreated) that are subject to rigorous quality control testing and are guaranteed to be RNase-free. DEC treatment is the most commonly used method for eliminating RNAS contamination from water, buffers, and other solutions.
A common concern that researchers have is the sweet, “fruity” aroma detected after autoplaying DEPC-treated solutions. The ethanol can combine with trace amounts of carboxylic acid to produce volatile esters, which give off this characteristic smell.
The amine groups tend to react with and “sop up” the DEC, making it unavailable for inactivating Cases (see Technical Bulletin 178). Unlike DEC, which does not inactivate Cases introduced post-treatment, RNA secure reagent–treated solutions can be reheated to help eliminate new contaminants.
While contaminating RNAS can result in a failed experiment, it is often difficult and time-consuming to determine which solution or piece of equipment is responsible. In the RNaseAlert Lab Test Kit procedure, an optimized RNA oligonucleotide, double-labeled with both fluorescent and quenching moieties, is introduced as a target for any contaminating RNAS.
The same technique can also be easily adapted for detecting RNAS contamination in tips, tubes, glassware, or any other surface. Cases can be introduced into RNA samples during RNA isolation (e.g., when small amounts of Cases are carried over into the preparation) or during normal day-to-day use, which inevitably leads to repeated opening/closing of sample tubes and insertion of possibly contaminated pipe tips.
Samples are detected by their fluorescence, and the signals are translated into electropherograms or into gel-like images (data not shown). Assessing the integrity of poly(A) RNA samples can be accomplished by performing a northern analysis, using a probe against a housekeeping gene such as GAP DH, cyclophilin, or beta-actin.
Since the RNA sample is resolved by size on a denaturing gel, degradation manifests as a smear below the full-length message. Absence of a band/smear at the expected full-length message size or observation of a band/smear at the bottom of the gel is an indication that the RNA is very severely degraded.
Degraded RNA samples are unsuitable for northern analysis, RACE protocols, or full-length CDA library construction. However, unless severely degraded, samples may not be affected in reverse transcription real-time PCR (RT-qPCR) analysis, if small amplicons are being analyzed.
The traditional method for combating Cases in enzymatic reactions such as in vitro transcription, reverse transcription, and translation is to use human placental ribonuclease inhibitor (also known as RNAS Inhibitor Protein, RI or PRI). A potential problem with the inhibitor is that it might be contaminated with the very Cases that it is designed to inhibit, due to copurification of nucleases.
Extended incubation of contaminated preparations of this protein has the potential to slowly release nuclease into enzymatic reactions. It is recommended as a superior alternative to placental ribonuclease inhibitor (RI or PRI) in any enzymatic reaction.
We also offer the RNA secure Reagent, a nonenzymatic alternative for use in reactions such as in vitro transcription, RT-qPCR, and translation. Unlike DEC, which does not inactivate Cases introduced post-treatment, RNA secure reagent–treated solutions can be reheated to help eliminate new contaminants.
Laboratory surfaces, such as bench tops, centrifuges, and electrophoresis equipment, should be assumed to be contaminated with Cases, since they are usually exposed to the environment. These surfaces get contaminated due to the presence of bacterial and fungal spores present in many laboratory environments.
Likewise, dead cells shed from human skin can also lead to contamination of exposed surfaces. Nitrogen RNaseZap™ Wipes, which are towelettes presoaked with the RNAS Zap reagent, are particularly convenient for decontaminating pipettes, bench tops, and other surfaces.
In lieu of immediate processing, liquid nitrogen is often used to rapidly freeze tissues after harvest to minimize RNA degradation. However, freezing tissue in liquid nitrogen and subsequent processing is not always convenient, especially if large numbers of samples need to be preserved.
Small amounts of Cases that may copurify with isolated RNA can compromise downstream applications. RNAS inhibitors are commonly used as a precautionary measure in most enzymatic manipulations of RNA to keep such contaminants in check.
If this procedure has been used, we recommend a Proteins K treatment followed by a phenol-chloroform extraction to eliminate all traces of RNAS prior to subsequent reactions. The presence of trace amounts of RNAS can compromise RNA integrity, even if the samples are stored frozen in an aqueous environment.
After the addition of RNA secure solution, simply heat the sample at 60 °C for 10 minutes to inactivate any Cases. It contains 1 mm sodium citrate, which is an efficient creator of divalent cations, and has a relatively low pH (~6.4) that minimizes RNA base hydrolysis.