Working with RNA

When working with RNA:


Nucleases (RNases) can destroy RNA and ARE EVERYWHERE, so you should:


Prepare an RNAse free working environment. Wear gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin. Change gloves periodically.

Lay a clean bench liner down in your bench and work over that. Have all tubes labeled and ready before use. You have to WORK FAST.

Dedicate a separate set of solutions for RNA work.

Wash glassware, mortar, pestles and spatulas, with 0.1% diethylpyrocarbonate solution (DECP treated water) over night and wrap in aluminum foil. Bake in oven at 180-250C for 8Hs or more before using. AUTOCLAVING IS NOT SUFFICIENT

Gel boxes and combs and plastic ware cannot be bake so wash with DECP treated water for 24 hs.

DEPC is a very useful reagent for making buffers and equipment RNase free (except Tris!). DEPC results in a covalent modification of nucleases rendering them inactive

Use aerosol tips for all procedures.

Clean your pipettes with Rnase AWAY (invitrogen).

Use RNAfree microcentrifuge tubes. Handle centrifuge tubes as little as possible, avoiding touching the inside of the lids.


The most common method used to assess the integrity of total RNA is to run an aliquot of the RNA sample on a denaturing agarose gel stained with ethidium bromide (EtBr). Intact total RNA run on a denaturing gel will have a sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication  that the RNA is completely intact. Run Also A RNA marker

The concentration of RNA can be determined by mixing 2 µl sample with 98 µl of DEPC-treated water and measuring the absorbance at 260 and 280 nm. Pure RNA should have an A260/A280 ratio of 2.0 or higher.


How to make DECP treated water


Mix 800ml of water with 0.8ml of DECP (in fridge 1 F1) Stir mix over nigh with a magnetic stirrer in the fume hood. Autoclave for an hour. DECP is VERY TOXIC (Take appropriate precautions when handling; e.g., always wear gloves and handle under a fume hood)


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