Mullins Molecular Retrovirology Lab

  • Department of Microbiology
  • School of Medicine
  • University of Washington
University of Washington/Fred Hutch Center for AIDS Research

mRNA Isolation with Qiagen OligoTex Kit

A. Reagents:

For mRNA isolation

- Binding Buffer OBB

  • 20 mM Tris-Cl pH 7.5, 1 M NaCl, 2 mM EDTA, 0.2% SDS

- Oligotex Suspension

  • 10% (w/v) in 10 mM Tris-Cl pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% NaN3 (Qiagen cat.no. 79000)

- Wash Buffer OW2

  • 10 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA

- Elution Buffer OEB

  • 5 mM Tris-Cl pH 7.5

- Small spin columns

  • RNase-free spin columns (Qiagen cat.no. 79523)

For mRNA precipitation

- 4M LiCl

- Linear acrylamide (5 mg/ml)

- 100% EtOH

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

B. Protocol:

1. Dilute no more than 250 µg total RNA in 250 µl ddH2O.

Add 250 µl OBB (pre-heated at 70°C).

Add 15 µl Oligotex (pre-heated at 37°C).

Mix by pipetting up and down.

Incubate at 70°C 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 µl OW2 by pipetting up and down.

Transfer to spin column in clean 1.5ml tube.

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

Transfer spin column to clean 1.5ml tube.

Add 400 µl OW2.

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

Transfer to spin column in clean 1.5 ml tube.

Add 100 µl OEB (pre-heated at 70°C).

Resuspend Oligotex by pipetting up and down.

Spin 14,000x g for 1 minute.

Save eluted mRNA on ice.

Optional: resuspend Oligotex with supernatant from step 7 and transfer to 1.5 ml 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

  1. LiCl added to 0.2 M (1:20)

  2. 20 µg linear acrylamide (carrier)

  3. 2.5 volumes 100% EtOH.

Store at -70°C.