Shirley Zhu/ total RNA labeling for Corning array
Fluorescent Probe Labeling for Microarray hybridization
-RT setup
total RNA 50-70 ug
Anchored oligo dT primer 4-5 ug
DEPC/H2O up to 15.4 ul
-Vortex and quick spin
-Incubate 70ºC for 10 min (Program: hyb 70C)
-Chill on ice for 1-2 min, quick spin and keep on ice
-RT Reaction for each individual tube: make a master mix of the first three reagents:
5X superscript II first strand buffer 6 ul
0.1 M DTT 3 ul
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 3 ul
Superscript II (BRL) 2 ul
Total volume: 30ul
-Mix well by tabbing gently and spin down
-Incubate at 42ºC for 1 hour. Qui (Program: hyb1)
-Add another 1 ul of Superscript II for RT booster and mix gently and spin down
-Incubate at 42ºC for 1 hour (Program: hyb1)
-Then cool on ice and add 3 ul of 0.5M EDTA stop solution, mix gently and quick spin
-Pool Cy3 and Cy5 samples to a microcon ( Option: pool all Cy3 sample together first, then add 30 ul to each 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), 20 ul of Human Cot1 DNA, 2ul of 10ug/ul polyA RNA, and 2 ulof 5ug/ul tRNA, mix and spin for 12 min at 12K at RT. Concentrate to a volume of less than 32 ul.
-Invert centricon into a new tib(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 32 ul
-Add 6.8 ul 20XSSC, and mix well
-Then add 1.2 ul 10%SDS to probe ( wipe exess 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.
-Prewarm hybridization chambers at this time with arrays inside( at 37C in slide warmer)
-Spin sample for 5 min
-And keep sample at 37 C
-Place 39 ul of probe onto array under a 22mm X 40 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 10% SDS
500 ml of 2X SSC/0.03% SDS 50 ml 1.5 ml
500 ml of 2X SSC 50 ml
500 ml of 1X SSC 25 ml
500 ml of 0.2 X SSC 5 ml
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