《Trizol法提RNA》doc版.doc

RNA sample treatment before Realtime- PCR protocolPart 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 SEPARATION2.1. Incubate the homogenized samples for 5 minutes at 15 to 30C 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 30C for 2 to 3 minutes. 2.3. Centrifuge the samples at no more than 12,000 g for 15 minutes at 2 to 8C. 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 PRECIPITATION3.1. 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 30C for 10 minutes and centrifuge at no more than 12,000 g for 10 minutes at 2 to 8C. 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 8C.5. REDISSOLVING THE RNA5.1. Dry the RNA pellet (air-dry), sometime need to use tips, open the cap and lay down the tube.5.2. Dissolve RNA in RNase-free water(90 l), and incubating for 10 minutes at 55C to 60C. 5.3. Stored at -70C.Part 2. DNase Digestion of RNA before RNA Cleanup(Base on QIAGEN the RNase-Free DNase Set, Cat. no. 79254):Note:Do not vortex the reconstituted DNase I. DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Store at 20C for up to 9 months. Thawed aliquots can be stored at 28C for up to 6 weeks. Do not refreeze the aliquots after thawing.Procedure 1. Mix the following in a microcentrifuge tube: _87.5 l RNA solution (contaminated with genomic DNA) 10 l Buffer RDD 2.5 l DNase I stock solution Make the volume up to 100 l with RNase-free water. The reaction volumes can be doubled if necessary (to 200 l final volume).2. Incubate on the bench top (2025C) for 10 min.Part 3. RNA Cleanup(Base on QIAGEN RNeasy Mini Kit, Cat. no. 74106):Procedure1. Add 350 l Buffer RLT, and mix well.2. Add 250 l ethanol (96100%) to the diluted RNA, and mix well by pipetting. Donot centrifuge. Proceed immediately to step 3.3. Transfer the sample (700 l) to an RNeasy Mini spin column placed in a 2 mlcollection tube (supplied). Close the lid gently, and centrifuge for 15 s at 8000 x g (_10,000 rpm). Discard the flow-through.*4. Add 500 l Buffer RPE to the RNeasy spin column. Close the lid gently, andcentrifuge for 15 s at _8000 x g (_10,000 rpm) to wash the spin column membrane. Discard the flow-through.5. Add 500 l Buffer RPE to the RNeasy spin column. Close the lid gently, andcentrifuge for 2 min at _8000 x g (_10,000 rpm) to wash the spin columnmembrane.6. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied),and discard the old collection tube with the flow-through. Close the lid gently, andcentrifuge at full speed for 1 min.7. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add3050 l RNase-free water directly to the spin column membrane. Close the lidgently, and centrifuge for 1 min at _8000 x g (_10,000 rpm) to elute the RNA.Part 4. RT (Reverse Transcription)(Base on Promega Reverse Transcription System, Cat. no. A3500 )1. Add reaction component into a tube and mix well.Component (put it on ice)AmountMgCl24l25mMReverse Transcription 10X Buffer2 l10mMdNTP Mixture2 lRecombinant RNasin Ribonuclease Inhibitor0.5 lout of -20C just before useAMV Reverse Transcriptase (High Conc.)0.5 lMust add after add buffer, out of -20C just before use,15