(Contributed by S. Ghoshroy, edited 11/15/2011 by DLR)
Preparation of material:
Read the following set of instructions before you plan to extract RNA. Solutions, and glassware should be ready before you take the samples out of the freezer for extraction. Before preparing solutions or starting extractions, metal scoops and spatulas, a mortar and pestle, a few beakers, Erlenmeyer flasks, and bottles should be baked at 275° C for 2 to 3 h. Treating solutions and water with 0.1% diethyl pyrocarbonate (DEPC) can reduce RNAse contamination. Compounds containing amine groups (e.g. Tris) cannot be DEPC and must be made using ultrapure stocks and DEPC water. Plasticware (blue (100-1000μl) and white (20-200μl) pipette tips, preloaded boxes, test tubes, etc.) untouched by humans should be autoclaved.
DEPC-treated water can be generated by treatment with 0.1% DEPC for 1 hour at 37ºC and autoclaving for 15-20 at 15 psi. For more information about working with RNA, we recommend reading “How to win the battle with RNase” in Sambrook and Russell, Molecular Cloning, Volume 1. 782-7.85.
Preparation of Solutions.
- Prepare 70% EtOH for RNA use with DEPC-treated water in RNase-free bottle or centrifuge tube.
- Preparation of solution D (denaturing solution) [Sambrook and Russel: Molecular Cloning. Volume 1, 7.4-7.8]:
- 4M Guanidinium thiocyanate
- 25mM Sodium citrate. 2H2O
- 0.5% (w/v) Sodium lauryl sarcosinate
- 0.1M β-mercaptoethanol.
- Add 47.28g of Guanidinium thiocyanate in 50 mL H2O
- (Solution D is very caustic. Wear appropriate gloves, a laboratory coat, and eye protection when preparing, handing, or working with this solution)
- 5 ml of 10% sarcosyl
- 3.3 ml of 0.75M sodium citrate pH 7
- Bring the volume to 100 ml with DEPC-treated H2O
- [Add β-mercaptoethanol to an aliquot of solution D before each use]
RNA extraction protocol
- Add liquid nitrogen to empty mortar and pestle, let it evaporate.
- Transfer all of the frozen tissue to a mortar containing liquid nitrogen and pulverize the tissue using a pestle. The tissue can be kept frozen during pulverization by addition of liquid nitrogen.
- Transfer the pulverized tissue to a polypropylene snap cap tube (falcon tubes), containing 3 ml of solution D. Add 22μl of β-ME for 3ml of solution D.
- 100 mg of tissue: 3 mL solution D. (Sambrook)
- 100 mg of cells: 1mL solution D (Lab protocol).
- 0.3 ml of 2M NaOAc pH 4. Mix.
[stock =3 M NaOAc. Use 0.2mL of 3M NaOAc pH 4- following the lab protocol]
- 3 mL of phenol. Mix. (This should be around pH 6.0 – we use OmniPur Phenol (6705) without the addition of the supplied Tris:EDTA buffer)
- 0.6 ml of chloroform: isoamyl alcohol (IAA) (49:1). (Prepare before you start extraction.)
- [Note: To minimize contamination by DNA trapped at the interface, avoid taking the lowest part of the interface.]
- [Important: Pellets are easily lost. Decant the supernatant into a fresh tube. Do not discard it until the pellet has been checked.]