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.

  • Department of Microbiology
    School of Medicine
    University of Washington
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