RNA Cleanup and DNase Digestion
(Qiagen RNeasy Mini Kit, #74104, RNase-Free DNase Set, #79254)
Before Starting:
a. Prepare DNase I stock solution:
o Dissolve the solid DNase I (1500 Kunitz units) in 550 l of RNase-free water.
Mix gently. Do not vortex.
o Divide the stock solution into single use aliquots 10 l, and store at -20°C in Box
7 for up to 9 months.
o Thawed DNase I stock solution aliquots can be stored at 2-8oC for up to 6 weeks.
Do not refreeze aliquots after thawing.
b. Prepare Buffer RLT
o Add 10 l -Mercaptoethanol to 1 ml of Buffer RLT
o If buffer RLT forms a precipitate, warm to re-dissolve.
c. Add 44mL ETOH to Buffer RPE.
d. Thaw RNA on ice.
Procedure (perform steps at room temperature):
1. Adjust 15 g (or less) of the RNA sample to a volume of 100 l with RNase-free water.
(If you add more than 15 g to the column it is likely to clog.) Add 350 l Buffer RLT,
and mix.
2. Add 250l 100% ethanol to the diluted RNA, and mix by pipetting. Do not centrifuge.
3. Apply the sample (700 l) to an RNeasy mini column placed in a 2 ml collection tube.
Close lid and centrifuge for 15 sec at >10,000 rpm. Discard the flow through and
collection tube.
4. Transfer the RNeasy column into a new 2 ml collection tube. Pipette 350 l Buffer RW1
into the RNeasy mini column, close lid and centrifuge for 15 sec at >10,000 rpm. Discard
the flow-through without allowing column to come in contact with the liquid.
DNase Digestion:
5. Add 70 l Buffer RDD (at 4oC) to 10 l DNase I stock solution. Mix by gently inverting
tube. Spin down liquid.
6. Pipette the DNase I incubation mix (80 l) directly onto the RNeasy gel membrane in
column and incubate at room temperature for 15 min.
7. Pipette 350 l buffer RWI into the RNeasy mini column, close lid and centrifuge for 15
sec at >10,000 rpm. Discard the flow-through.
Buffer RPE wash and Elution
8. Pipette 500 l of diluted Buffer RPE to the RNeasy column. Close lid and centrifuge for
15 sec at >10,000 rpm. Discard the flow through.
9. Add another 500 l Buffer RPE to the RNeasy column. Close lid and centrifuge for 2 min
at > 10,000 rpm. Discard the flow through and the collection tube.
10. Transfer the RNeasy column into a new 2 ml collection tube. Centrifuge at full speed for
1 min to dry the membrane.
11. Transfer the column to a 1.5ml microcentrifuge tube. Pipette 30 l of RNase-free water
directly on the membrane. Centrifuge at full speed for 1 min to elute the RNA.
Updated 01/16/15, LM
12. Prepare 250ng of sample (you can go as low as 150 ng see picture below) + 2 L of blue
loading dye (6X) + DEPC water qs for 12L in RNase-free tubes. Load onto gel. Run at
80V / 400mA for one hour. Look for 28S and 18S bands. (28S bands (4.8 kb) should be
more intense than 18S bands (1.9 kb), and there should be no smear under 28S bands.)
13. Store the eluted liquid at -80oC or move on to RT.
Updated 01/16/15, LM