Trizol法提RNA.doc

RNA sample treatment before Realtime- PCR protocol Part 1. RNA Extraction (Base on Invitrogen TRIZOL? Reagent, Cat. No. 15596-026) Procedure 1. HOMOGENIZATION 1.1. Aspirate the medium from the wells. 1.2. Add TRIZOL? Reagent 1ml/well, and passing the cell lysate several times through a pipette. (Base on 6wells plate) Note: The amount of TRIZOL Reagent added is based on the area of the culture dish (1 ml per 10 cm2) and not on the number of cells present. An insufficient amount of TRIZOL Reagent may result in contamination of the isolated RNA with DNA. 1.3. Transfer the homogenate solution to fresh tubes (1.5ml Eppendorf tube) 2. PHASE SEPARATION 2.1. Incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. 2.2. Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes. 2.3. Centrifuge the samples at no more than 12,000× g for 15 minutes at 2 to 8°C. Note: Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. 3. RNA PRECIPITATION 3.1. Transfer the colorless upper aqueous phase to a fresh tube. (1.5ml Eppendorf tube) 3.2. Use 0.5 ml of isopropyl alcohol (per 1 ml of TRIZOL Reagent used for the initial homogenization). Incubate samples at 15 to 30°C for 10 minutes and centrifuge at no more than 12,000 × g for 10 minutes at 2 to 8°C. 4. RNA WASH Remove the supernatant. Wash the RNA pellet once with 1 ml 75% ethanol(use the nuclease free water dilute), adding at least 1 ml of 75% ethanol (per 1 ml of TRIZOL Reagent used for the initial homogenization). Mix the sample by vortexing and centrifuge at no more than 7,500 × g for 5 minutes at 2 to 8°C. 5. REDISSOLVING THE RNA 5.1. Dry the RNA pellet (air-dry), sometime need t