Shirley Zhu/ mRNA labeling
Fluorescent Probe Labeling for Microarray hybridization
-RT setup
mRNA
2-4 ug
Anchored oligo dT primer
4-5 ug
DEPC/H2O
up to 15.6ul
-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
6 ul
0.1 M DTT
3 ul
50 X dNTPs
0.7ul
(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)
1.7ul
Total volume:
30ul
-Mix well (vortex and spin down)
-Incubate at 42ºC for 1 hour (Program: hyb1)
-Add another 1 ul of Superscript II for RT booster and mix (vortex and spin down)
-Incubate at 42ºC for 1 hour (Program: hyb1)
-Degrade mRNA with 1.5 ul of 1 M NaOH/2 mm EDTA (vortex and spin down)
-65 C for 8 min. (do NOT go TOO long here) (Program: hyb-65C-8)
-Add 15ul of 0.1 M HCL for each (vortex and spin down)
-Pool Cy3 and Cy5 samples, Cy3 adds to Cy5 in a 1.5 ml RNase free tube
-Then Add 450ul of 1X TE (pH 7.4) with 20ul of Human COT DNA (1ug/ul) to the
sample; mix well (vortex and spin down)
-Add the about 600ul TE with cDNAs to microcon YM 30 filter
-Spin in Eppendorf centrifuge until volume equal about 20ul (9-11’) at 10K rpm,
-Add 500 ul of 1X TE to the microcon YM 30 filter
- Spin at 10K rpm for 9-11 min until volume equal about 20ul,
-Add 500 ul 1X TE again, spins until volume less than 28.2ul,
BE VERY CAREFUL TO NOT SPIN THE SAMPLE DRY!!
-Invert microcon, recover labeled samples by spinning 2 min.
-Adjust sample volume to 28.2ul by TE and transfer to a new micro tube
-To the 28.2ul of combined Cy3+Cy5 sample, add the following:
Yeast tRNA
1ul (10mg/ml)
Poly A DNA
2ul (10mg/ml)
20XSSC
5.9ul
10%SDS
0.9ul
Final volume:
38ul
-1-
Shirley Zhu/ mRNA labeling
-Mix well; avoid bubbles (vortex and spin down)
-Heat sample at 100 ºC for 2 min 42ºC for 20–30 min, (Program: hyb2)
-Meanwhile, get and set up the necessary number of Hyb chambers and get coverslips
ready, and get arrays ready, write down array No. –sample No.- chamber No (optional)
- Take samples out of 42 C, spin down and bring to RT (wait 2-5 minutes)
-Add 38ul of probe onto the center of the array while NOT actually touching the array
face with the pipette tip
-Quickly and gently place the coverslip onto the array face (use two fine forceps)
-Add about 2ul of 3XSSC in 8-10 drops onto the top 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