Ambion miRNA Labeling Protocol
(thaw all non-enzyme reagents to room temp for two hours prior to reaction)
Poly A tail miRNA
1. Add 250 ng miRNA to two 1.5 ml tubes. Dry down in speed vac on medium heat
for about 15 min.
2. Resuspend pellet in 3 µl nuclease free water.
3. To each tube add:
Diluted positive control miRNA
(1 µl in 499 µl nuclease free water)
1 µl
2X poly(A) Polymerase Reaction Buffer
10 µl
25 mM MnCl2
2 µl
10X Amine-NTP Mix
2 µl
Poly(A) Polymerase
2 µl
Mix by flicking tube gently and microfuge.
4. Incubate reaction @ 37ºC for 2 hours.
Tailing Rxn Clean-up
Preheat Elution Solution to 95ºC
1. To each tube add 10 µl miRNA carrier and 350 µl miRNA binding/ wash buffer
(ethanol added). Mix by vortexing and incubate at room temp. for 5 min.
2. Pipette mixture onto miRNA labeling cartridge and centrifuge 10,000xg for 15
sec. Discard flowthrough.
3. Pipette 300 µl miRNA binding/wash buffer to cartridge and centrifuge 10,000xg
for 15 sec. Discard flowthrough and repeat 1X.
4. Dry filter by centrifuging @ 10,000xg for 1 min.
5. Transfer cartridge to collection tube. Add 15 µl 95ºC Elution Solution directly to
filter. Incubate @ 65ºC for 10 min.
6. Centrifuge @ 10,000xg 15 sec. to collect tailed miRNA
7. Add 15 µl Elution solution to cartridge and and incubate 65ºC for 10 min.
Centrifuge 10,000xg for 1 min to collect all tailed miRNA.
8. Dry sample in vacuum concentrator.
Dye Coupling Rxn
1. Add 7 µl nuclease free water to each sample and vortex briefly to resuspend.
2. Add 16 µl DMSO to Cy 3 and Cy5 Post labeling Reactive Dye (vortex and spin
down, keep in dark until used)
3. To each tube of 7 µl sample add:
9 µl Coupling buffer
4 µl Cy3 or Cy5 in DMSO
Vortex briefly and spin down.
4. Incubate in dark at room temp for 1 hr.
5. Add 4.5 µl 4 M Hydroxylamine, vortex and microfuge, incubate @ room temp in
dark for 15 min
Labeling Reaction Clean-Up
NOTE: Here you will run the Cy3 labeled and Cy5 labeled sample that are going on the
same array through the same tube
1. Add 350 µl miRNA Binding/Wash buffer to each labeling reaction and vortex
briefly to mix.
2. Incubate at room temp in dark for 5 min.
3. Pipette into labeling cartridge and centrifuge @ 10,000xg for 15 sec. Add second
sample to same tube and centrifuge @ 10,000 xg for 15 sec.
4. Add 300 µl Binding/Wash buffer to labeling cartridge and centrifuge @ 10,000xg
for 15 sec. Discard flowthrough. Repeat 1X
5. Centrifuge 1 min @ 10,000xg to dry out filter.
6. Place cartridge into new collection tube. Add 22 µl 95ºC Nuclease free water.
Incubate at 65 ºC for 10 min at room temp.
7. Centrifuge @ 10,000xg for 1 min to collect labeled sample in tube.
8. Use labeled sample within a half hour.
9. Proceed to hyb protocol.