P-TABM-461 - P-TABM-461
RNA ISOLATION with TRIzol Reagent 1. Caution : When working with Trizol Reagent use gloves and eye protection. Avoid contact with skin or clothing. Use in a chemical hood.TRIzol is toxic in contact with skin and if swallowed. Causes burns. After contact with skin, wash immediately with plenty of detergent and water. If you feel unwell, seek medical advice. Contains Phenol and other components. 2. Material and reagents required : - Dry ice - Liquid nitrogen - Chloroform - Isopropanol - 75 % Ethanol (in DEPC-water) - RNase-free water : Add 100 µl DEPC (diethylpyrocarbonate) to 100 ml nanopure water. Let stand overnight and autoclave. - RNase-free tips, eppendorf-tubes (2 ml and 1.5 ml),... - TRIzol reagent 3. Precautions for preventing RNase contamination : - Always wear gloves. Skin often contains bacteria and molds that can contaminate a RNA preparation and be a source of RNases. - Use a new bag of tips or a bag with tips only used for RNA-work. - Use gloves to fill boxes with tips. - Use sterile, disposable plasticware reserved for RNA work to prevent cross-contamination with RNases from shared equipment. - Always use DEPC-treated water during the preparation. 4.METHOD : 4.1. Grinding of the plant tissue : Grind the plant tissue under liquid nitrogen to a fine powder (in an 2 ml eppendorf-tube). Allow the liquid nitrogen to evaporate. Do not allow the sample to thaw (Keep the samples on dry ice !). Note : Incomplete grinding of the starting material will lead to reduced RNA yield. 4.2. Homogenization of the plant tissue : Homogenize tissue samples in 1 ml TRIzol Reagent (per 50-100 mg of tissue). Add the trizol in 2 times: at first add 200 and grind the tissue in the solution, and then add 800 ml. Mix the sample by vortexing until the tissue is dissolved. From now on you can work at room temperature ! Incubate the homogenized samples for 5 minutes at room temperature. 4.3. Phase separation : Add 200 µl chloroform per 1 ml TRIzol Reagent. Cap the tubes securely and shake by hand for 15 seconds. Incubate at room temperature for 2 - 3 minutes. Centrifuge the samples for 15 minutes at 11 000 rpm at 4 °C. 4.4. RNA precipitation Transfer the aqueous phase to a fresh Rnase- free tube. Add 500 µl isopropanol per 1 ml TRIzol Reagent. Incubate the samples for 10 minutes at room temperature. Centrifuge for 10 minutes at 11000 rpm and at 4 °C. 4.5. RNA wash Remove the supernatant. Wash the RNA pellet with 1 ml of 75 % ethanol per 1 ml TRIzol Reagent. Centrifuge the sample for 5 minutes at 11 000 rpm and at 4 °C. Dry the pellet for 10 minutes at room temperature (air-dry). 4.6.Redissolving the RNA Add 40 µl DEPC-water. Incubate the samples for 10 minutes at 65 °C in a thermoblock. Leave the samples on ice for overnight (at 4 °C) (or shorter). Centrifuge for 10 minutes at 11 000 rpm. Carefully remove the supernatant to a new Rnase-free tube. Mesure the concentration with the spectro-fotometer (OD 260 / 280) or check 2 µl RNA on an agarosegel (1.2 % agarose/ 0.5 x TBE buffer prepared with DEPC-H2O).