Shirley Zhu_total RNA_for Oligo arrays
Amino-allyl Indirect Labeling for HEEBO arrays
(This protocol basically followed the protocols from Brown Lab
http://cmgm.stanford.edu/pbrown/protocols/RTaminoAllylCoupling.html
http://cmgm.stanford.edu/pbrown/protocols/Direct_Label_Protocol1.html
And combine the protocol From NCI http://www.riedlab.nci.nih.gov/publications/cDNA%20indirect%20%20labelling.pdf .
Some modifications have been made. This modified protocol was developed by Shirley Zhu
and has room for refinement.)
First Day:
RT setup
total RNA(after DNase treatment and purification) 30ug
Anchored oligo dT primer (Operon, HPLC purified) 1ul(4-5ug/ul)
Random Hexamer (Amersham, Cat#:27-2166-01) 1ul(4-5ug/ul)
Doping control (SFGF, DCV2.1) 1ul
(MJ Cy5 mix with samples, MJ Cy3 mix with UHR Ref.)
H2O(RNase free) up to 18.4ul
-Vortex and quick spin
-Incubate 70ºC for 10 min
-Chill on ice for 10 min
-RT Reaction for each individual tube: (make a master mix )
5X superscript III first strand buffer 6ul
0.1 M DTT 3ul
50X aa-dUPT 0.6ul
(50ul of 100 mM dATP/dCTP/dGTP,
25ul of 100 mM dTTP,
50ul of 50 mM aa-dUTP (Ambion 8439)
Store t -20 in 30ul aliquots)
Superscript III (Invitrogen) 2ul
Total volume: 11.6ul
-Add to RNA: 18.4ul
Total volume: 30ul
-Mix well by tabbing gently and spin down
-Incubate at 42ºC for 2 hour.
Hydrolysis
-Incubate at 95°C for 5 minutes
-Add 13ul 1N NaOH, 1ul .5M EDTA (pH is now ~13)
-Incubate at 67°C for 15 minutes
-Neutralize with 50ul 1M HEPES (USB Cat: 16925, pH 7.3)
(Samples can be stored at 4°C or -20°C for a few days)
Second day:
Cleanup probes with Qiagen Minelute Reaction Cleanup (28204)
-Add 300ul buffer ERC to columns
-Add 30ul 3M Na-Acetate pH 5.2(Sigma, Cat# S-7889)
-Add sample, mix, spin@ 14K rpm for 1 min
- Discard flow-through, add 750ul buffer PE, spin @14K rpm for 1 min
- Discard flow-through, spin for 1 min to dry column
-Place column in clean 1.5 ml tube, add 12.5 ul 10mM Na-Phosphate pH 8.5
(To prepare 10mM Na-Phosphate solution pH 8.5:
Volume
0.22 uM filtered 1M Na2HPO 98 ul
0.22 uM filtered 1M NaH2PO 2 ul
Water 9.9 ml)
-spin @14K rpm for 1 min
-Repeat once
-should get about 25ul, adjust by 10mM Na-Phosphate pH 8.5, if less than 25ul.
Coupling
(The dyes should not be exposed to water prior to coupling and should be
used immediately after they are resuspended in DMSO. Reactions should be carried
out in the dark as the Cy dyes are light sensitive. )
-Resuspend monofunctional NHS-ester Cy3 or Cy5 dye (Amersham RPN5661) in 25ul
DMSO (JTBaker 9224-01).
- Mix with 25ul of probe solution and incubate in dark at room temp for 90 min.
(If you use one dye pack for multiple samples, it is important to maintain 50% v/v DMSO in the coupling reaction. I am using one dye pack for 2 samples, so resuspend Cy3/Cy5 in 50ul, 25ul for each.)
Removal of unincorporated dyes with QIAquick PCR Purification Kit, CAT#28104
(Some modifications have been made)
-Pool Cy3 and Cy5 coupling reactions, get total volume: 100ul
-Add 10ul of 3M Na-Acetate pH 5.2(Sigma, Cat# S-7889) to the coupling reactions (optional)
-Add 500ul of PB buffer to the coupling reactions and mix well
-Apply 600ul to column, spin for 1min, and discard flow-thru
-Add 750ul of Buffer PE and spin for 1min @ 14K rpm
-Repeat once
-Add 750ul of 80%ETOH and spin for 1 min @14K rpm
-Spin additional 1 min to dry column
-Place column into a new tube
-Add 18.5ul buffer EB, let sit 1 min, spin 1 min @ 14K rpm
-Repeat
-adjust volume to 36.68ul by H2O.
Hybridization
-Transfer probe to a micro tube to adjust volume to 36.68ul by H2O
-Add (can make Master MIX)
2ul of 10ug/ul Human Cot1 DNA,
2ul of 10ug/ul polyA RNA,
2ul of 10ug/ul yeast tRNA
1.32ul of HEPES
-Add 9.35ul 20XSSC, and mix well
1.65ul 10%SDS to probes (wipe excess SDS off exterior of pipette tip)
-Make total volume of 55ul
-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.
-Spin sample for 10 min
-And keep sample at RT for 15-20 min
Array Preparation with Hybex and Lifter Slips:
-Clean LifterSlips with clean water followed by 100% EtOH rinse.
-Dry LifterSlips with clean dry air. Dust off arrays as well.
-Wet the pad in the tops of Hybex racks
-Lay the lifter slips on each slide
-One at a time, slowly pipette 55uL probe onto the array, by pipetting the solution onto the slide, just adjacent to one corner of the LifterSlip.
-Place each array in a separate Hybex Rack, and screw the Hybex Chamber together.
-Once two chambers have been done, place in a Hybex block and set to 65oC.
-Hybridize at 65oC for 18-40 hours.
Third day: (working at ozone free area)
Wasing and Scanning
-Unscrew chamber
- Gently allow coverslip to fall off in 2X SSC/0.03%SDS at room temperature and transfer slides from Hybex rack to a slide rack
-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.
-Transfer slides to another rack in 2XSSC washing buffer shaking with an orbital shaker
for 5 min at Room temperature
-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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