EXTRACCIN DE RNA

Materials:

Chloroform Isopropyl alcohol 75% ethanol (in DEPCtreated water) RNase-free water or 0.5% SDS Centrifuge and rotor capable of reaching up to 12,000 g Polypropylene microcentrifuge tubes Homogenizer. Pestles washed in DEPCtreated H2O Water bath or heat block (5560C) Pipettor

Methodology: Tissues 1. Add 1 mL TRIzol Reagent per 50100 mg of tissue sample. 2. Homogenize sample using a glass-Teflon or power homogenizer. Note: Process or freeze tissue samples immediately upon collection. Phase separation 1. Incubate the homogenized sample for 5 minutes at room temperature to permit complete dissociation of the nucleoprotein complex. 2. Add 0.2 mL of chloroform per 1 mL of TRIzol Reagent used for homogenization. 3. Shake tube vigorously for 15 seconds. 4. Incubate for 23 minutes at room temperature. 5. Centrifuge the sample at 12,000 g for 15 minutes at 4C. Note: The mixture separates into a lower red phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The upper aqueous phase is ~50% of the total volume. 6. Remove the aqueous phase of the sample. Avoid drawing any of the interphase or organic layer into the pipette when removing the aqueous phase. 7. Place the aqueous phase into a new tube and proceed to the RNA Isolation Procedure.

RNA precipitation 1. Add 0.5 mL of 100% isopropanol to the aqueous phase, per 1 mL of TRIzol Reagent used for homogenization. 2. Incubate at room temperature for 10 minutes. 3. Centrifuge at 12,000 g for 10 minutes at 4C. Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube. 4. Proceed to RNA wash. RNA wash 1. Remove the supernatant from the tube, leaving only the RNA pellet. 2. Wash the pellet, with 1 mL of 75% ethanol per 1 mL of TRIzol Reagent used in the initial homogenization. 3. Vortex the sample briefly, then centrifuge the tube at 7500 g for 5 minutes at 4C. Discard the wash. 4. Vacuum or air dry the RNA pellet for 510 minutes. Do not dry the pellet by vacuum centrifuge. Note: Do not allow the RNA to dry completely, because the pellet can lose solubility. 5. Proceed to RNA resuspension. RNA resuspension 1. Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution (2050 L).. Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions. 2. Incubate in a water bath or heat block set at 55 60C for 1015 minutes. 3. Proceed to downstream application, or store at 70C.

Existen otros mtodos para purificar RNA? S, pero presentan algunas desventajas como el no ser posible utilizar la muestra obtenida para un procesamiento simultneo. Por mencionar otros mtodos tenemos el que se basa en cido tricloroactico y acetona, o el basado en sales de guanidina. La componente principal es un agente desnaturalizante potente que desnaturalize las RNasas. Qu hacer contra las RNasas? No usar buffers contaminados (RNasas no se remueven por autoclave) Usar pipetas libres de RNasas, por ejemplo que no hayan sido utilizadas para preparacin de plsmidos. Usar guantes y limpiarlos cada vez que se toca un aparato u otra cosa sin descontaminar Usar buffers en pequeas alcuotas y desechar despus del primer uso Tratar las soluciones con 0.1% de DEPC por almenos 1 h a 37 C y luego autoclavear Tratar el instrumental que se va a utilizar con DEPC o algn producto que inactiva RNasas (RNaseZap) Usar tubos y puntas certificados en estar libres de RNasas.

Dietilpirocarbonato: uso y cuidados. Es un agente altamente reactivo alcalino, que inactiva por la etoxiformilacin de grupos histidil. Debido a que modifica indiscriminadamente protenas y RNA, no puede ser utilizado durante la extraccin, y es incompatible con ciertos buffers como el Tris. Al degradarse genera pequeas cantidades de etanol y CO2 los cuales incrementan la fuerza inica y bajar el pH de una solucin. Las modificaciones que este agente causa pueden ser revertidas por el tratamiento con 0.5 M de hidroxilamina a pH neutro.