3. Add 1.4 mL 100% ethanol and immediately transfer each sample to an RNeasy Midi column.
4. Centrifuge the samples for 5 min at 4500 Â g and RT.
5. Transfer the flow-through to a RNase-free 50 mL Corex tube
kept on ice.
6. Add 2.5 mL RPE buffer onto each column for the first wash of
the column.
7. Centrifuge the samples for 5 min at 4500 Â g and RT.
8. Transfer the flow-through of the first wash to the previously
used Corex tube.
9. Add 2.5 mL RPE buffer onto the column for the second wash
of the column.
10. Centrifuge the samples for 5 min at 4500 Â g and RT.
11. Transfer the flow-through of this second wash to the Corex
tube. The LMW RNA is now in the flow-through/wash aliquots that were combined in the Corex tube (for HMW
RNA recovery, see Note 10).
12. Add 10 mL cold isopropanol to each LMW RNA sample.
13. Close the Corex tubes with parafilm and invert seven times.
14. Leave the samples overnight at À20
C for the small RNAs to
precipitate.
15. The next day: Centrifuge the samples for 45 min at 24,000 Â g
and 4
C.
16. Discard the supernatant carefully with a pipette.
17. Add 10 mL cold 75% ethanol to each sample for washing.
18. Centrifuge the samples for 15 min at 24,000 Â g and 4
C.
19. Discard the supernatant (see Note 11), air-dry the RNA pellets
at RT for about 20 min, and then heat the tubes to 65
C for
5 min.
20. Resuspend the dried RNA pellets in 30 μL DEPC-treated
water, preheated to 65
C.
21. Quantify the LMW RNA using the Nanodrop device. Samples
can be stored at À20
C.
3.4 Northern Blot
Procedure
A schematic overview of the northern blot procedure is depicted in
Fig. 2.
1. Transfer up to 10 μg LMW RNA from each sample to a clean
1.5 mL microfuge tube, adjusting all samples to the same total
volume using DEPC-treated water (see Note 12).
2. Prepare a size standard using synthetic RNA oligonucleotides
and the low-range ssRNA ladder. Bring this mix to the same
Genome-Scale and Northern Blot Analyses of siRNAs
399
4. Centrifuge the samples for 5 min at 4500 Â g and RT.
5. Transfer the flow-through to a RNase-free 50 mL Corex tube
kept on ice.
6. Add 2.5 mL RPE buffer onto each column for the first wash of
the column.
7. Centrifuge the samples for 5 min at 4500 Â g and RT.
8. Transfer the flow-through of the first wash to the previously
used Corex tube.
9. Add 2.5 mL RPE buffer onto the column for the second wash
of the column.
10. Centrifuge the samples for 5 min at 4500 Â g and RT.
11. Transfer the flow-through of this second wash to the Corex
tube. The LMW RNA is now in the flow-through/wash aliquots that were combined in the Corex tube (for HMW
RNA recovery, see Note 10).
12. Add 10 mL cold isopropanol to each LMW RNA sample.
13. Close the Corex tubes with parafilm and invert seven times.
14. Leave the samples overnight at À20
C for the small RNAs to
precipitate.
15. The next day: Centrifuge the samples for 45 min at 24,000 Â g
and 4
C.
16. Discard the supernatant carefully with a pipette.
17. Add 10 mL cold 75% ethanol to each sample for washing.
18. Centrifuge the samples for 15 min at 24,000 Â g and 4
C.
19. Discard the supernatant (see Note 11), air-dry the RNA pellets
at RT for about 20 min, and then heat the tubes to 65
C for
5 min.
20. Resuspend the dried RNA pellets in 30 μL DEPC-treated
water, preheated to 65
C.
21. Quantify the LMW RNA using the Nanodrop device. Samples
can be stored at À20
C.
3.4 Northern Blot
Procedure
A schematic overview of the northern blot procedure is depicted in
Fig. 2.
1. Transfer up to 10 μg LMW RNA from each sample to a clean
1.5 mL microfuge tube, adjusting all samples to the same total
volume using DEPC-treated water (see Note 12).
2. Prepare a size standard using synthetic RNA oligonucleotides
and the low-range ssRNA ladder. Bring this mix to the same
Genome-Scale and Northern Blot Analyses of siRNAs
399