Amino-allyl Reverse Transcriptase Labeling Protocol and Hybridization Prep Diaz Lab
(25Sep03)/ Updates: JR 15Feb05; ED 08Jul05
1. Reverse transcription reaction
Annealing with random hexamers (total volume 16.5 µl):
Concentration
amplified RNA
1-5 µg
Random Hexamers
3µg/µl
RNAse-free Water
a. Incubate @ 70ºC for 10 min
b. Chill on ice
Reverse transcription (Invitrogen):
Master mix
µl (for each RXN)
5X first strand buffer
6
50X aa-dUTP/dNTPs
0.6
DTT
3
RNase Out
1
Water
1.4
Total Volume per RXN
12
µl
2
Up to 16.5 µl
50X aa-dUTP/dNTPs
0.1 M dATP 10µl
0.1M dGTP 10µl
0.1M dCTP 10µl
0.1M aa-dUTP 6µl
0.1M dTTP 4µl
Incubate @ RT for 10 min
Add 1.5 µl Superscript II, gentle mix (total RXN volume should be 30 µl)
Incubate @ 42ºC for 2 hr
2. Hydolysis
Add to each RXN: 10 µl 1 M NaOH
10 µl 0.5 M EDTA
Incubate @ 65ºC for 15 min. (prepare 1 mL tubes for transfer and label Zymo columns)
Neutralize with Zymo column binding buffer- 1 mL
3. Clean-up (remove Tris to prevent monofunctional NHS-ester Cy dye coupling to
free amine of the buffer)
Mix binding buffer & RT RXN well, then transfer 550 µl to Zymo column
Centrifuge @ 9,500 rcf for 1 min
Aspirate flowthru. Add remaining 450 µl of RT RXN to column
Centrifuge @ 9,500 rcf for 1 min
Add 200 µl wash buffer to each column
Centrifuge @ 9,500 rcf for 1 min
Repeat wash with 200 µl wash buffer and spin again
Transfer to fresh tube (1.5 mL w/out cap), spin @ 16,000 rcf for 1 min to dry column
Elute with 0.1 M sodium bicarbonate pH 9.0 (made fresh)- i.e., add 6 µl, incubate @ RT
for 5 min., spin @ 16,000 rcf for 1 min., add 6 µl, spin again
Total volume = 10-11 µl. Take 1 µl for gel, take 1 µl for Nanodrop (DNA Technologies
Core Facility)
Freeze rest or proceed to coupling.
4.
a.
b.
c.
d.
Coupling
Use freshly resuspended tubes of Cy dye (split 3 ways)
Add 3.5 µl DMSO to each Cy tube. Pipet rigorously to resuspend all clumps
Take 1 µl dye & add to RT tube. Mix and spin down if necessary.
Incubate @ RT in dark for 1.5 hrs.
5. Clean-up
To remove unincorporated Cy dyes, proceed with Zymo column clean-up:
Add 500 µl binding buffer to each tube of RT couple with dye
Mix well and transfer to Zymo column
Centrifuge @ 9,500 rcf for 1 min
Add 200 µl wash buffer to each column
Centrifuge @ 9,500 rcf for 1 min
Repeat wash with 200 µl wash buffer and spin again
Transfer to fresh tube (1.5 mL w/out cap), spin @ 16,000 rcf for 1 min to dry column
Elute with prewarmed to 37ºC Qiagen buffer EB-add 6 µl, incubate @ RT for 5 min
Centrifuge @ 16,000 rcf for 1 min
Should yield 5 µl
Use 1 µl for Nanodrop for quality control
Mix 5 µl of sample (Cy5) and 5 µl of reference (Cy3)
6. Hybridization Prep
Prepare clean cover slips! Check to make sure they are flat before washing. Wash with
water and EtOH, wipe with Kimwipe, blow dry with compressed air.
Make 6 mL 3X SSC, 50% formamide for humidification chambers.
Hybridization buffer recipe:
Stock Solution
µl per slide
cDNA sample
10
1M Hepes, pH 7.8
1
Poly-A (20 µg/µl)
1
20X SSC
6
Formamide
20
10% SDS
2
(add separately last)
Total volume:
40
Prepare tubes, 1 per array, 10 µl of labeled cDNA- add hyb buffer in order above (pipet
SDS with care!)
Incubate probe @ 100ºC for 2 min
Spin down on nanofuge to collect and pipet to mix.
Apply to prepared microarray and place in humidification chamber (small black
microscope slide holder) containing two kimwipes saturated with 3X SSC, 50%
formamide at bottom of chamber.
Seal with sequencing tape
Hybridize O/N @ 50ºC
Proceed to microarray wash protocol.
Reagents:
aminoallyl-dUTP (Sigma cat. #A0410)
DNA Clean & Concentrator kit (Zymo cat. #D4005)
Random hexamers (Amersham, cat #RPN5661)
CyDye Post-labeling Reactive Dye Packs (Amersham cat #27-2166-01)
Superscript II (Invitrogen cat #18064-022)