CH 12/08
Arcturis PiciPure RNA isolation from LCM samples
(with changes and additional steps by Ben Matthews’ lab)
1. Add 50 l Extraction Buffer (XB) to the PCR tube cap (If samples are spread between
multiple tubes, combine all tissue samples in 50 l final volume)
2. Vortex right-side up and upside-down
3. Incubate at 42 degrees for 30 minutes
a. While incubating:
i. Pipette 250 l Conditioning Buffer (CB) onto the purification column
filter membrane
ii. Incubate at least 5 minutes at room temperature
iii. Centrifuge column at 16,000 x g for 1 minute
4. After the 30 minute incubation is complete, add 50 l of 70% EtOH (from the kit) into
the cell extract. Pipette up and down to mix. DO NOT CENTRIFUGE.
5. Transfer the cell extract/ETOH mixture to the pre-conditioned purification column.
6. Bind RNA to column by spinning for 2 minutes at 100 x g followed by a spin at 16,000
x g for 30 seconds to remove flow-through.
a. Do not empty collection tube at all throughout extraction, just let waste build up.
7. Add 100 l Wash Buffer 1 (W1) to the column and spin for 1 minute at 8,000 x g
8. Prepare a 40 l solution of DNAse using one of the following methods:
a. Peggy’s version: Combine 4 l of 10X Ambion Turbo DNA-free Buffer, with 35
l DEPC H20 and 1 l of Ambion DNA-free DNA DNAse enzyme (Cat no.
AM1907)
b. Arcturis version: Comine 5 l of Qiagen DNAse I stock solution (Cat no.
79254) to 35 l of Buffer RDD (comes with the DNAse kit)
9. Add this 40 l solution directly to the purification column membrane. Do not spin.
10. Incubate either:
a. Peggy’s version: 20 minutes at 37 degrees
b. Arcturis Version: 15 minutes at room temp
11. Add 40 l of Arcturis PicoPure Wash Buffer W1 to the column membrane
12. Spin for 15 seconds at 8,000 x g
13. Add 100 l of Wash Buffer 2 (W2) onto the column and centrifuge for 1 minute at 8,000
xg
14. Add another 100 l of W2 to the column and spin for 3 minutes at 16,000 x g.
15. Transfer purification column to a new 0.5ml tube (provided).
16. Pipette 11 l (recommended, but 30 l maximum) elution buffer (EB) directly onto the
membrane (gently touch tip of pipette to the surface of the membrane)
17. Incubate EB on column for one minute at room temp
18. Spin for 1 minute at 1,000 x g to distrubute EB in column
a. When spinning, place 0.5ml tube inside a capless 1.5 ml tube, and put a regular
1.5ml tube next to tube with column (see diagram in manual)
19. Spin for 1 minute at 16,000 x g to elute RNA. Store -80oC.