RNase contamination

Subsequent to DNA template linearization, the procedures for in vitro RNA transcription reaction are set up. It is essential to avoid RNase contamination by using gloves, sterile glassware, and water devoid of RNase activity [treated with 0.1% diethyl pyrocarbonate (DEPC) and autoclaved]. 

Perform electrophoresis, and visualize the mRNA sample by ethidium bromide staining. If the RNA band is smeared or not visible, possible causes may be degradation of mRNA by RNase contamination. 

This step is very important to avoid RNase contamination. 

All solutions used for reverse transcription must be free of RNase. If all solutions are prepared with RNase-free water in disposable plastics and no contact with RNase-contaminated spatula or pH probes has occurred, the solutions need not be treated any further. Otherwise, to decontaminate the solutions add diethyl pyrocarbonate (DEPC) (e.g., Sigma, St. Louis, MO) to the solution to a final concentration of 0.1%, shake, let sit overnight with loosened caps, and then autoclave for 15 min. Note that DEPC might be carcinogenic and that solutions containing Tris cannot be decontaminated with DEPC, but must be prepared with special caution to prevent any RNase contamination. 

Great care should be taken to ensure that all apparatus and chemicals are sterile and free from RNase contamination. Glassware should be incubated at above 150°C for at least 6 h. All solutions not containing amines should be treated with diethyl-pyrocarbonate (DEPC) and autoclaved. If solutions contain amines (Tris), RNase-free chemicals should be added to DEPC-treated distilled water. Gloves should be worn at all times and changed frequently. 

Microdissected cells for protein analysis may be stored at -80°C before extraction. Microdissected cells for DNA analysis may be stored desiccated at room temperature up to 1 week before extraction. Samples for RNA analysis should not be stored before extraction. Condensation in the microcentrifuge tube during storage may be a potential source of RNAse contamination. RNA extraction should be performed immediately after microdissection. Store extracted RNA samples at -80°C before amplification. 

RNA is relatively an unstable molecule and significantly more susceptible to degradation than DNA, mainly because of the presence of ribonudeases (RNases), which represent different enzymes that break down RNA molecules. RNases are very stable, do not require cofactors, are effective in very small quantities and are difficult to inactivate. RNase contamination can come from human skin and dust partides, which can carry bacteria and molds. They may be present as well in cell lysates as in the surrounding environment, consequently the isolation and analysis of RNA requires highly specialized techniques. 

In this case, the most common sources of RNase contamination are hands and dust or aerosol with microorganisms. Therefore, latex or vinyl gloves should be worn and frequently changed when handling reagents and RNA samples. Tubes containing samples or reagents should be kept closed wherever possible. Furthermore, it is also important to keep isolated RNA on ice. 

Endogenous RNases are another source of RNase contamination released by the disruption of the cells during extraction. These RNases can be inactivated using various inhibitors of RNases. Inhibitors of RNases are used to suppress the activity... 

Tissue sections are obtained from rats that are killed by decapitation. The brains (or other required tissue) are rapidly removed and frozen in isopentane that has been precooled (-30°C) on dry ice. Sections (14 pM) are cryostat-cut and mounted (two sections per slide) under conditions that minimize RNase contamination. Mounting sections close to the bottom of the subbed slides will conserve reagents required for double-label ISH. Multiple adjacent series of sections are obtained, depending on need. For example, it is advisable to take a series for each [ Sj-labeled riboprobe both alone and in combination with each appropriate digoxigenin-labeled riboprobe. Slide-mounted sections are sorted at -20°C (in cryostat or cold room) prior to the assay to obtain the required number of near adjacent series. Slides brought to room temperature cannot be... 

Whichever method of purification is chosen, other precautions to eliminate adventitious RNase contamination should also be employed, especially by the less practiced worker. Even the most experienced worker occasionally suffers from the degradation of RNA preparations associated with RNase contamination. This can be from many sources and even in some cases appear to be an act of God. When problems are encountered, or if you are not accustomed to working with RNA, the following precautions should be employed. 

Compared with DNA, more care has to be taken when working with RNA to prevent any RNase contamination. Use of gloves (hands are the major source of RNase), diethyl pyrocarbonate (DEPC)-treated solutions, disposable tubes, pipet tips from freshly opened bags, and RNasin (a potent ribonuclease inhibitor) in the labeling reaction, minimizes the risk of RNA degradation. 

PLL-coated slides Always wear gloves during this procedure. Place the RNase-free slide on clean aluminum foil or a similar surface that has been wiped with absolute alcohol to avoid RNase contamination. With a pencil, mark the slide on the side to be coated, as PLL is transparent and impossible to detect once applied. Apply a small drop (approx 5 xL) of PLL solution to the slides. Spread a thin film of PLL over the whole surface. Either use the same method as for making blood films, or appose the surface of two slides to spread the drops of liquid and then slide them apart. The PLL film dries quickly (1-2 min) and the tissue sections can be picked up onto the coated surface immediately afterwards (see Note 1). 

PLL-coated slides are best used immediately, but batches may be prepared and stored in the baked rack, wrapped in the aluminum foil to protect from dust and RNase contamination. PLL-coated slides can be stored at room temperature for up to 1 mo. 

When mRNA is the target, special care should be taken, and all the specimen handling procedures should be carried out using clean, disposable gloves and sterile instruments, in order to avoid RNase contamination. 

All the equipment used in this procedure can be cleaned with 10% peroxide solution and washed with copious amounts of sterile water to avoid RNase contamination. 

To avoid degradation of target mRNAs, all reagents must be free from RNase contamination until after hybridization. Wear gloves, use disposable plastics, and use autoclaved solutions where possible. 

It is very important to handle and store the digoxigenin-labeled riboprobes with care to avoid degradation by RNase contamination The probes should be stored at -80°C with an RNase inhibitor for long term use and can be stored at -20 C for daily use... 

To avoid potential RNase contaminations from other sources, especially from hands, clean gloves should be worn for all RNA manipulations. Water can be made RNase free by treatment with DEPC (0.1%), followed by autoclaving. 

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