RNAIsolationProtocol

RNA Isolation Protocol
(Revised 5-15-2003)

Stabilize RNA

Start with 15 ml E. coli Culture containing 7.5* 10⁹ cells (OD₆₀₀= 0.2 Dilute cells or scale up)

• Pipet 30 ml of RNAProtect Bacteria Reagent (Qiagen) into a 50ml polypropylene conical tube.

• Pipet 15 ml culture into the tube.  Mix immediately by vortexing for 5second.  Incubate for 5min at room temperature.

• Centrifuge for 10min at 5800g( 4500rpm for H-6000A rotor in SORVALL RC-3B centrifuge)

• Decant the supernatant, and leave tubes inverted on a paper towel for 10s.

• Freeze the pellet with EtOH/Dye Ice mix.

• The pellet can be stored at -20°C up to 2 weeks, or -70°C for up to 4 weeks.

Isolation RNA

• Dilute 2ul of Proteinase K into 300ul of Tissue and Cell Lysis solution for each sample.

• Resuspend cell pellet by the Lysis solution and mix thoroughly. Transfer mix to 1.5ml tube.

• Incubate at 65°C for 45min, and vortex every 15min.

• Place the sample on ice for 5min.

• Add 150ul of MPC protein Precipitation Reagent to 300ul of lysed sample and vortex mix for 10sec.

• Spin for 10min 4°C at max speed in a microcentrifuge. Transfer the supernant to a clean tube.

• Add 50ul of MPC protein Precipitation Reagent and repeat above step.

• Add 500ul isopropanol to the recovered supernatant, invert the tube 30-40 times.

• Pellet the RNA by centrifugation at 4°C for 10min in a microcentrifuge.

• Carefully pour off the isopropanol without dislodging the RNA pellet. Remove all of the residual isopropanol with a pipet. Air dry 10-15min.

Removal of contaminating DNA

• Dilute 10ul of RNAse-Free DNAse I up to 200ul with 1x DNAse Buffer for each sample.

• Completely resuspend the nucleic acid pellet in 200ul of DNAse I solution.

• Incubation at 37deg;C for 45min

• Add 200ul of 2x T and C lysis solution, vortex mix for 5 seconds

• Add 200ul of MPC reagent vortex mix 10seconds, place on ice 5min.

• Pellet the debris by centrifugation for 10min at 4°C, >10,000g in a microcentrifuge.

• Add 50ul of MPC reagent and repeat 5 and 6 (but this time spin 20min).

• Add 600ul isopropanol to the recovered supernatant, invert the tube 30-40 times.

• Pellet the RNA by centrifugation at 4°C for 10min in a microcentrifuge.

• Carefully pour off the isopropanol without dislodging the RNA pellet.

• Rinse with 75% EtOH (DEPC H₂O), being careful to not dislodge the pellet. Centrifuge 5min at 4°C

• Remove all of the residual EtOH with a pipet. Air dry 10-15min.

• Resuspend the nucleic acid in 52ul RNAse-free water.

Storage, Quantitation and Determination of Quality of RNA

• Electrophoresis on 1% Agarose gel with 1ul sample.

• Dilute 1ul sample to 100ul with TE(10mM Tris.HCl pH 8, 1mM EDTA), and measure A260 and A280 with a spectrophotometer.. Alternatively 1ul sample can be used to measure A260 and A280 on a Nanodrop ND-1000 Spectrophotometer without dilution.

• Concentration of RNA sample = 40 x A260 x 100 (Dilution factor) (ug/ml)

• A260/A280 Ratio = A260/A280, ranging from 1.7 to 2.1

• Add 100ul EtOH (2 volume) and 5ul 3M NaOAC (1/10 volume), store at -20°C

Regents

• RNAProtect Bacteria Reagent Qiagen

• MasterPure RNA Isolation Kit Epicentre

Note: This protocol was adapted from the original Qiagen and Epicentre protocols