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RNA protocols

RNA storage RNAase-free environment RNA quantitation and quality control  Comments

section mRNA purification on Dynabeads ds cDNA synthesis internal links Northern blot/hybridization

RNA storage

Traditionally, RNA is stored at -70°C, but there are no obvious reasons, why it should be unstable at -20°C.

High-quality RNA is stable: we do not recognize any degradation after 1 day incubation at 37°C in a buffer with Mg²⁺.

Safe way of transportation at ambient temperature is a salt/ethanol precipitate.

If RNA would be used only for electrophoresis it is possible to solubilize it in formamide (RNases do not work in formamide).

RNA is chemically unstable in alkali solutions (especially at high temperature) and in the presence of two-valent ions at high temperature. Products of degradation have phosphorilated 3' ends.






RNAase-free environment

The problem is, that RNases are (i) thermostable and (ii) can work without two-valent ions. So, two easy ways of struggle against DNases in solutions (autoclaving and presence of EDTA) do not work for RNases. The main way to avoid RNase contamination is clean work.




Normally, clean work is absolutely enough to keep the integrity of RNA. It is better not to use diethilpirocarbonate (DEPC) treated reagents, because DEPC-treatment may decrease activity of some enzymes.




Main sources of RNases in the laboratory:

dry RNase keep it in a poliethylene bag in a special place. Whenever possible use single-use aliquotes. Weight RNase under fume hood with all possible precautions. RNase A solutions keep in a special place, clean tubes with wet paper towels, clean gloves after work. bacterial media weight it under fume hood, do not pour water directly from water source. bacterial growth use freshly prepared steril buffers. sweat, tears, saliva do not cough and talk in front of tubes. sweat when it is hot, put a piece of paper towel on your hand under the glove; wash gloves regularly. skin work in gloves. Transfer RNA in a new tube, if you ocassionaly grasp tube cap with bare hand. dust use separate plastic for work with RNA. Keep racks with tubes and tips closed, clean table regularly.






RNA quantitation and quality control

RNA concentration may be determined both spetrophotometrically and by fluorophore staining.

DNA contamination is easily distinguishable by electrophoresis or by fluorescent staining: double-stranded DNA staines better than single-stranded RNA; it runs higher than ribosomal RNA;

The easiest way to check the integrity of total RNA: electrophoresis or capillary electrophoresis (Agilent). There is a special scale for RNA degradation. On practice, if you work accurately, RNA is always "highest rank" quality. RNA degradation scale is for preparation of RNA is already degraded sources (dead animals, patalogo-anatomy studies, etc.).

Electrophoresis:

• express electrophoresis: normal 1.5% gel, fresh TAE buffer. Quantitation (by comparison with sample with known quantity). Analysis of DNA-contamination. Control of RNA degradation.
• Formaldehyde-gel electrophoresis.
• use separate chamber for electrophoresis of RNA. Washing of dirty chamber:
     (i) wash with detergent,
     (ii) rinse with mQ,
     (iii) fill with 2% H₂O₂ for 15-30 min,
     (iv) rinse with mQ again.