Description Isolation of total cytoplasmic RNA using LiCl-Urea extraction and PK digestion – Auffray and Rougeon modified method RNA extraction suitable for RNases rich tissues Procedure Sacrifice animals by cervical dislocation. Dissect and remove tissues immediately and snap-freeze them in liquid nitrogen. Store snap-frozen tissues at -70°C for a short period. NB : Do not store too long, especially if tissue is RNase rich. Day 1 : Disrupt the frozen organ using mortar and pestle kept in liquid nitrogen, and transfer the powder to 14 ml tube containing LiCl-Urea solution (8 ml/ g of tissue). Add 50 µl SDS 20% per 2 ml LiCl-Urea solution. Homogenize carefully the powder in solution using a Polytron (PT1200) on ice. Clean Polytron probe between samples (in 50 ml tubes) : 1 time with SDS 1 % followed by 2 times in H2ODMPC, then 1 time in NaOH 0.5 N and rinse 2 times with H2ODMPC. Incubate overnight on ice (precipitation). Day 2 : Centrifuge 10 min at 13 000 g at 4°C, remove carefully the supernatant. Resuspend the pellets in the same volume of LiCl-Urea solution (without SDS) and incubate at least 30 min on ice. Centrifuge 10 min at 13 000 g at 4°C, remove carefully the supernatant. Resuspend the pellets in 400-750 µl PK buffer, and transfer in Eppendorf tubes. Incubate for 20 min at 37°C. Extract with phenol/ chloroform : Add ½ volume of phenol, vortex thoroughly, and add ½ volume of chloroform, vortex and centrifuge at 10 000 g for 10 min at 4°C. Transfer the aqueous phase (aqueous 1) in fresh tube and extract with one volume of chloroform, vortex and centrifuge at 10 000 g for 10 min at 4°C. Transfer the aqueous phase in a fresh tube. Add one volume of PK Buffer (without PK) on the organic phase, vortex thoroughly and centrifuge at 10 000 g for 10 min at 4°C. Transfer the aqueous phase 2 in fresh tube and reextract with one volume of chloroform. Pool the aqueous phases, and precipitate RNA with 2.5 volumes of 100 % ethanol. Gently mix and precipitate at least 2 h at -20°C (overnight precipitation is recommended for small samples). Centrifuge at 13 000 g at 4°C, remove the supernatant carefully and wash with 1 volume of 75 % Ethanol, centrifuge as before and dissolve the pellet in sterile water (H2ODMPC)or formamide (20-200 µl), depending on expected yield and downstream applications. Quantitate by UV absorbance at 260 nm and check the ratio of UV absorbance at 260 and 280 nm. 17 Check an aliquot of the RNA by ethidium agarose gel electrophoresis. Store the RNA at -70°C. Recipes Reagents : All reagents must be made with sterile MilliQ water, treated with DMPC (add DMPC 0.1%, incubate 2h à 37°C, and autoclave to inactivate DMPC). LiCL-Urea Buffer : Sodium acetate pH 5.3 10 mM; urea 6 M; LiCl 3 M PK Buffer : Tris-HCl 10 mM pH 8 ; EDTA 2 mM pH 8 ; NaCl 200 mM ; SDS 0.5 % ; 200 µg/ml proteinase K (Invitrogen, RNase free). Prepare it freshly at RT or SDS/EDTA will precipitate. Phenol, Chloroform, Ethanol 100 %, Ethanol 75 % Supplies Mortar and pestle. Polytron or equivalent. Centrifuge Sorvall or Jouan with fixed angle rotor. (责任编辑:泉水) |