mRNA Isolation with Qiagen OligoTex Kit
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 Âµg), Midi (70042, 250 Âµg-1mg) and Maxi (70061, 1-3mg)
1. Dilute no more than 250 Âµg total RNA in 250 Âµl ddH2O.
Add 250 Âµl OBB (pre-heated at 70Â°C).
Add 15 Âµl Oligotex (pre-heated at 37Â°C).
Mix by pipetting up and down.
Incubate at 70Â°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 Âµl 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 Âµl OW2.
Spin at 14,000x g for 1 minute. Discard flow-through.
Transfer to spin column in clean 1.5 ml tube.
Add 100 Âµl OEB (pre-heated at 70Â°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
LiCl added to 0.2 M (1:20)
20 Âµg linear acrylamide (carrier)
2.5 volumes 100% EtOH.
Store at -70Â°C.