mRNA Isolation Protocols --(1)  (with Qiagen Oligotex kit) -- (2) 

A. Reagents:

For mRNA isolation

Binding Buffer OBB

20 mM Tris-Cl pH7.5, 1 M NaCl, 2 mM EDTA, 0.2% SDS

Oligotex Suspension

10% (w/v) in 10 mM Tris-Cl pH7.5, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% NaN3.

Wash Buffer OW2

10 mM Tris-Cl pH7.5, 150 mM NaCl, 1 mM EDTA

Elution Buffer OEB

5 mM Tris-Cl pH 7.5

Small spin columns

RNase-free spin columns

For mRNA precipitation

4M LiCl

Linear acrylamide (5 mg/ml)

100% EtOH

Oligotex mRNA kits Mini (70022, <250¦Ìg), Midi (70042, 250¦Ìg-1mg) and Maxi (70061, 1-3mg)

B. Protocol:

Dilute no more than 250 ¦Ìg total RNA in 250 ¦Ìl ddH2O.

Add 250 ¦Ìl OBB (pre-heated at 70C).

Add 15 ¦Ìl Oligotex (pre-heated at 37C).

Mix by pipetting up and down.

Incubate at 70C for 3 minutes.

Incubate at RT for 10 minutes.

Spin at 14,000 x g for 2 minutes.

Remove supernatant and save for second extraction (step 20).

Resuspend pellet in 400 ¦Ìl OW2 by pipetting up and down.

Transfer to spin column in clean 1.5 ml tube.

Spin at 14,000 x g for 1 minute. Discard flow-through.

Transfer spin column to clean 1.5ml tube.

Add 400 ¦Ìl OW2.

Spin at 14,000 x g for 1 minute. Discard flow-through.

Transfer to spin column in clean 1.5ml tube.

Add 100 ¦Ìl OEB (pre-heated at 70C).

Resuspend Oligotex by pipetting up and down.

Spin 14,000 x g for 1 minute.

Save eluted mRNA on ice.

Optional: resuspend Oligotex with supernatant from step 7 and transfer to 1.5ml tube. Repeat extraction from step 4. Combine eluted mRNA.

Read OD260/280 and calculate concentration.

Run 50-200 ng mRNA on Bioanalyzer.

Ethanol precipitate with

LiCl added to 0.2 M (=1:20)

20 ¦Ìg linear acrylamide (carrier)

2.5 volumes 100% EtOH.

Store at -70C.