RNA Isolation Protocol

互联网2013-09-06

837

RNA Isolation Protocol
(Revised 5-15-2003)

Stabilize RNA

Start with 15 ml E. coli Culture containing 7.undefined 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 H₂ O), 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

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