Shirley Zhu/ total RNA labeling for Corning arrays
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
-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.
-Add another 1 ul of Superscript II for RT booster and mix gently and spin down
-Incubate at 42ºC for 1 hour
-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 microcon, 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 ul of 5ug/ul yeast tRNA, mix and spin for 12 min at 12K at RT. Concentrate to a volume of less than 32 ul.
-Invert microcon into a new tub (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 probes (wipe excess SDS off exterior of pipette tip using latex glove before adding to probes). Mix by tapping lightly and quick spin to minimize bubbles.
-Denature probes by heating for 2 min at 100C then incubate at 37C for 20-25 min.
-Pre warm 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 40 ul 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 cover slip 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 cover slip 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 cover slip 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
Pre-hybridization of Corning cDNA arrays
1. Mark boundaries of array on back of slide using diamond scriber. Array will become invisible after post-processing.
2. Fill bottom of humid chamber with 100 ml H2O of 65C
3. Place arrays face down over H2O and cover chamber with lid.
4. Rehydrate until array spots glisten. Allow spots to swell slightly but not run into each other.
5. Snap-dry each array (DNA side up) on a 70-80C inverted heat block for 3 seconds.
6. UV crosslink DNA to glass with Stratalinker set for 60 mJ. (Set display to "600", which is 600 x 100 uJ.)
7. Incubate arrays at 55C for 1 hour in Prehyb solution ( keep at 55C):
3X SSC, 0.1% SDS, 0.1mg/ml BSA
8. Rinse arrays for 5 minutes in ddH2O at RT
9. Boil arrays in ddH2O for 2 minutes
10. Plunge arrays into 95% ETOH
11. Centrifuge at 500 rpm for 5 minutes
12. Use same day or store at RT and use within 2 days.
- 1 -