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定量PCR RNA extraction 1. Homogenize tissue samples in 1ml of TRIzol Reagent per 50-100mg of tissue using a glass-Teflon or power homogenizer. 2.Incubate the homogenized samples for 5 minutes at 15-30℃ to permit the complete dissociation of nucleoprotein complexes. 3.Add 0.2ml of chloroform per 1ml of TRIzol Reagent.Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30℃ for 2 to 3 minutes. 4.Centrifuge the samples at no more than 12000×g for 15 minutes at 2 to 8℃. 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. The volume of the aqueous phase is about 60%of the volume of TRIzol Reagent used for homogenization. 5.RNA precipititation Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein desired. 6. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5ml of isopropyl alcohol per 1ml of TRIzol Reagent used for the initial homogenization. 7.Incubate samples at 15 to 30℃ for 10 minutes and centrifuge at no more than 12000×g for 10 minutes at 2 to 8℃ .The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. 8.RNA wash Remove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1ml of 75% ethanol per 1ml of TRIzol Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 7500×g for 5 minutes at 2 to 8℃. 9.Redissolving the RNA At the end of theprocedure, briefly dry the RNA pellet (air-dry or vacuum-dry for5-10 minutes). Do not dry the RNA by centrifugation under vacuum. It is important not to let the RNA pellet dry cpmpletely as this will greatly decrease its solubility. Partially dissolved RNA samples with sterilized water. DNase treatment of bulk samples DNAses from