EXTRACTION OF PROTEIN, RNA, AND DNA USING THE TRIZOL® PROTOCOL (LAB 3/4/08) TRI Reagent® is a complete and ready-to-use reagent for the isolation of total RNA or the simultaneous isolation of RNA, DNA and proteins from samples of human, animal, plant, yeast, bacterial and viral origin. We will use it to extract these macromolecules from bee larvae (kindly provided by Dr. Hranitz). Today we will focus on isolating and characterizing the total RNA fraction, saving the others for later analysis. TRI Reagent combines phenol and guanidine thiocyanate in a mono-phase solution to facilitate the immediate and most effective inhibition of RNase activity. A biological sample is homogenized or lysed in TRI Reagent and the homogenate is separated into aqueous and organic phases chloroform addition and centrifugation. RNA remains exclusively in the aqueous phase, DNA in the interphase and proteins in the organic phase. RNA is precipitated from the aqueous phase by addition of isopropanol, washed with ethanol and solubilized. DNA and proteins are sequentially precipitated from the interphase and the organic phase with ethanol and isopropanol, washed with ethanol and solubilized in water. NOTE: RNAse contamination is a common problem. Liberally treat the work area with RNASEAway™. Each bench will have 1 liter of DEPC-treated water from which all of your solutions are to be made. DEPC (diethyl pyrocarbonate) is an RNAse inhibitor. Because TRI Reagent® is toxic, and your hands are contaminated with RNAses, use gloves for all manipulations. STAGES OF RNA ISOLATION OVERVIEW: 1. 2. 3. 4. 5. HOMOGENIZATION: Bee larvae PHASE SEPARATION: homogenate + 0.2 ml chloroform. RNA PRECIPITATION: aqueous phase + 0.5 ml isopropanol. RNA WASH: 1 ml 75% ethanol. RNA SOLUBILIZATION: water. DETAILED PROTOCOL: 1. HOMOGENIZATION: Weigh larva. If less than 75 mg, combine two in the same 1.5 ml tube. Add 1 ml TRI Reagent® and thoroughly grind using a homogenizer, pipette tip, or Bead-Beater™ . Use 1 ml of TRI Reagent® per 50150 mg tissue (i.e., use 1 ml for this experiment). 2. PHASE SEPARATION: a. Incubate the homogenate for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Spin at top speed for 1m to remove debris. Transfer liquid supernatant to a new tube. b. Add 0.2 ml chloroform/ ml TRI Reagent®. Vortex for 15 seconds. c. Incubate @ rmT for 10m. d. Spin @ top speed @ 4ºC X 15m. e. Following centrifugation, the mixture separates into a lower red phenolchloroform phase, an interphase and the colorless upper aqueous phase. RNA remains exclusively in the aqueous phase whereas DNA and proteins are in the interphase and organic phase. The volume of the aqueous phase is about 60% of the volume of TRI Reagent used for homogenization 3. RNA PRECIPITATION a. Transfer the aqueous phase to a fresh tube and save the interphase and organic phase at 4ºC for subsequent isolation of DNA and proteins. b. Precipitation of RNA. 1) Add 0.5 ml isopropanol/1.0 ml TRI Reagent® (i.e., 0.5 ml isopropanol). 2) Mix. 3) Incubate@RmT X 10 m. 4) Spin @ top speed @ 4ºC X 8m. NOTE: RNA precipitate forms a gelatinous white pellet on the side of the tube. It is often invisible before centrifugation. 3. RNA WASH a. Remove the supernatant and add 1 ml 75% ethanol to resuspend pellet. b. Mix vigorously. c. Spin @ ½ top speed @ 4ºC X 5m. NOTE: If the RNA pellet accumulates on the side of the tube and has a tendency to float, re-spin the pellet at top speed X 5 m. 4. RNA SOLUBILIZATION a. Pour off the ethanol wash and briefly air-dry the pellet (3 - 5 m). b. Dissolve RNA with DEPC-treated water by passing the solution a few times through a pipette tip and incubating for 10-15 minutes at 55-60 C. 4. RNA QUANTITATION AND VISUALIZATION a. Quantitate your sample spectrophotometrically by reading@A260. b. Visualize your total RNA on a 0.8% TAE agarose gel. c. Load 1 µg total. Heat to 65ºC X 10 m, and then add RNA Gel Loading Dye (~2X). d. Use the 1kb marker as your size standard. NOTE: rinse your gel box with RNASEAway™, use DEPC–treated water to make 1X TAE and agarose. STORE THE REMAINDER OF YOUR REACTIONS (PROTEIN AND DNA FRACTIONS) @ -20ºC IN YOUR SAMPLE BOX.