Shirley Zhu/ total RNA labeling
Fluorescent Probe Labeling for Microarray hybridization
-RT setup
total RNA
50-70ug
Anchored oligo dT primer
4-5 ug
DEPC/H2O
up to 15.4ul
-Incubate 70ºC for 10 min (Program: hyb 70C)
-Chill on ice for 1-2 min.
-RT Reaction for each individual tube: make a master mix of the first three reagents:
5X superscript II first strand buffer
6ul
0.1 M DTT
3ul
50 X dNTPs
0.6ul
(500 uM dATP/dCTP/dGTP, 200 uM dTTP)
Add these reagents to each tube individually:
Cy3 (pink, for Ref. RNA, always) or
Cy5 (blue, for samples, always) dUTP
3ul
Superscript II (BRL)
2ul
Total volume:
30ul
-Mix well (vortex and spin down)
-Incubate at 42ºC for 1 hour (Program: hyb1)
-Add another 1ul of Superscript II for RT booster and mix (vortex and spin down)
-Incubate at 42ºC for 1 hour (Program: hyb1)
-Then cool on ice and add 3ul of 0.5M EDTA stop solution, mix gently and quick spin
-Pool Cy3 and Cy5 samples to a microcon
-Wash1: Add 450ul of 1X TE (pH 7.4) mix well by inverting tubes, spin 11 min at 12K g
at RT,
-Wash2: pour off flow through, add 500 ul 1X TE (pH7.4) to each centricon, spin 11 min
at 12Kg at RT
-Wash3: pour off flow through, add 450 1X TE (pH7.4), 20ul of Human Cot1 DNA, 2ul
of 10ug/ul polyA RNA, and 2ul of 5ug/ul tRNA, mix and spin for 12 min at 12Kg at RT.
Concentrate to a volume of less than 32ul.
-Invert centricon into a new tube (lid cut off) and spin for 2 min at 12K g at RT to recover
the probe.
-Transfer probe to a microtube to adjust volume to 32ul
-Add 6.8ul 20XSSC, and mix well
-Then add 1.2ul 10%SDS to probe ( wipe excess SDS off exterior of pipette tip using
latex glove before adding to probe ). Mix by tapping lightly and quick spin to minimize
bubbles.
-Denature probe by heating for 2 min at 100C then incubate at 37C for 20-25 min.
-Pre-warm hybridization chambers at this time at 37C in slide warmer
-Spin sample for 5 min
-And keep sample at RT for about 20 min.
-1-
Shirley Zhu/ total RNA labeling
-Place 40ul of probe onto array under a 22mm X 60 mm glass cover slip; Add about 2ul
of 3XSSC in 8-10 drops onto the middle axis of the array coverslip away from the actual
array border for hydration purposes.
-Assemble the Hyb chamber with the array slide in it, turn the screws gently till you can’t
move them anymore and place into a 65ºC water bath overnight
Next day:
-Pullout the Hyb chamber and dry off the excess H2O
-Disassemble the Hyb chamber, and quickly place the slides tilted with the coverslip
slightly down into a slide rack in a washing chamber that contains 2XSSC/0.03 % SDS
(RT), repeat this, individually for each array, one at a time, until all are done; and wait
until the coverslip falls off.
-Then transfer the slides to 2X SSC/0.03%SDS at 65C, shake the slide holder up and
down vigorously for 5 min, making sure slides never out solution.
-Wash slides in 2XSSC for 5 min, same as above or with a shaker
-Wash slides in 1XSSC for 5 min, same as above
-Wash slides in 0.2XSSC for 5 min, twice
-Spin slides down in centrifuge at 500 rpm for 5 min (prepare everything in advance, so
transfer of slide rack to holder is very quickly done)
- Clean box needed to transport slides to scanner
-Scan immediately!
Washing buffer:
20X SSC stock solution
500 ml of 2X SSC/0.03% SDS
500 ml of 2X SSC
500 ml of 1X SSC
500 ml of 0.2 X SSC
50 ml
50 ml
25 ml
5 ml
-2-
10% SDS
1.5 ml