RNeasy Protocol for RNA Cleanup
With DNase treatment
Kits: RNeasy Mini Kit (Qiagen #74104)
RNase-Free DNase Set (Qiagen #79254)
Before starting, make fresh Buffer RLT with β-Mercaptoethanol (β-ME)
Add 10 λ β-ME per 1 mL Buffer RLT
Need 350 λ Buffer RLT/sample
Before starting, make fresh RNase-Free DNase (If DNase treating!)
Dissolve solid DNase I (1500 Kunitz units) in 550 λ RNase-free water
Mix gently, DO NOT VORTEX
Store in aliquots at -20 °C for up to 9 months (refrigerate for up to 6 weeks)
1. Bring RNA volume up to 100 λ with RNase-free water. Add 350 λ Buffer RLT (with βME). Mix thoroughly.
2. Add 250 λ ethanol (96-100%) to diluted RNA. Pipet up and down to mix. Do not spin.
3. Apply the sample (700 λ) to an RNeasy mini column in a 2 mL collection tube.
Centrifuge at max speed (13,200 rpm) for 15 seconds. Discard flow-through.
4. Add 350 λ Buffer RW1 onto column. Centrifuge 15 seconds at 13,200 rpm. Discard
flow-through.
5. In a separate tube, add 10 λ DNase I stock solution (reconstituted) to 70 λ Buffer RDD.
Mix gently (DO NOT VORTEX).
6. Add DNase I incubation mix (80 λ from Step 5) directly onto membrane. Let sit for 15
minutes at room temperature.
7. Add 350 λ Buffer RW1 onto column. Centrifuge 15 seconds at 13,200 rpm. Discard flow
through.
8. Transfer column into new 2 mL collection tube (supplied). Add 500 λ Buffer RPE
(DILUTED WITH ETHANOL). Centrifuge for 15 seconds at 13,200 rpm. Discard flow
through.
9. Add another 500 λ Buffer RPE to column. Centrifuge 2 min at 13,200 rpm. Discard flow
through and tube.
10. Transfer to a new 1.5 mL collection tube. Centrifuge for an additional 1 min.
11. To elute, transfer column to new 1.5 mL collection tube (supplied). Pipet 30-50 λ RNasefree water directly onto the membrane .LET SIT FOR 2 MINUTES! Centrifuge 1 minute
(longer for higher yield) at 13,200 rpm.
12. To increase yield, repeat the elution step (#11) with a small amount of RNase-free
water. Centrifuge for 1 min at 13,200 rpm.