- Rice 3'Tiling Microarray Hybridization
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For the synthesis of double strand cDNAs, Superscript Double-Stranded cDNA Synthesis
Kit(Invitrigen, U.S.A.) was used.
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Combine 10µl(10µg) of total RNA with 1µl of oligo dT
primer(100µM) and heat to 70 °C for 10 minutes. Briefly spin tubes in a
microcentrifuge, and place the reaction(s) in an ice. Add 4µl of 5X First
Strand Buffer, 2µl of 0.1M DTT, 1µl of 10mM dNTP mix and 2µl of SuperScript
enzyme. Place sample(s) in a incubator of 42°C for 1 hour. Add 91µl of DEPC-Water,
30µl of 5X Second Strand Buffer, 3µl of 10mM dNTP mix, 1µl of 10U/µl DNA
ligase, 4µl of 10U/µl DNA Polymerase I and 1µl of 2U/µl RNase H to the
first strand reaction(s). Incubate at 16°C for two hours and add 2 µl of 5
U/µl T4 DNA polymerase to each reaction. To remove RNA strand, add 1µl
of 10 mg/ml RNase A(Amresco, U.S.A.) solution to the tubes and incubate
sample(s) at 37°C for 10 minutes. To precipitate cDNA, add 180µl of
phenol/chloroform and centrifuge at maximum speed for 5 minutes. Transfer
upper phase into new tube, add 16µl of 7.5 M Ammonium Acetate and 326µl of
cold ethanol. Centrifuge at maximum speed for 20minutes and remove
supernatant. Add 500µl of 80% ethanol and centrifuge again at maximum speed
for 5 minutes. Remove supernatant and dry the pellet. Rehydrate samples
with 20µl of water. For the synthesis of C3-labeled target DNA fragments,
mix 1µg of double strand cDNA with 40µl(1OD) of Cy3-9mer
primers(Sigma-Aldrich, U.S.A.), make the volume of 80µl with deionized
water and then, heat to 98°C for 10 minute. Add 10µl of 50X dNTP mix (10mM
each), 8µl of deionized water, 2µl of Klenow fragment(50 U/µl, NEB, U.S.A.)
and incubate at 37°C for 2 hours. Finally, stop the reaction by addition of
10µl of 0.5M EDTA. Precipitate DNA fragments by adding 11.5µl of 5M NaCl
and add 110µl of isopropanol to each tube. Vortex and incubate at room
temperature for 10 minutes. Centrifuge at maximum speed for 10 minutes.
Remove supernatant with pipette. Add 500µl of 80% ethanol and centrifuge
again at maximum speed for 5 minutes. Remove supernatant and dry the
pellet. Rehydrate samples with 25µl of water. Determine the concentration
of each sample uing spectrophotometer.
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Hybridization and Scanning
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Mix 13µg of DNA with 19.5µl of 2X hybridization
buffer(Nimblegen, U.S.A.) and make the volume of 39µl with deionized water.
Hybridize the sample solution with microarray (Nimblegen, U.S.A.) using
MAUI chamber(Biomicro, U.S.A.) at 42°C for 16 hours. After the
hybridization, remove chip from MAUI Hybridization Station and immerse in
the shallow 250ml Wash I (Nimblegen, U.S.A.) at 42 °C. Agitate the chip in
Wash I for 10-15 seconds and transfer the slide into the second dish of
Wash I. Incubate 2 min with agitation. Transfer slide rack into dish of
Wash II. Incubate 1 minute with agitation. Transfer slide rack into dish of
Wash III. Incubate for 15 seconds with agitation. Remove slide rack from
Wash III and spin dry in a centrifuge for 1 minute at 500g. Store the dried
array in a dark desiccator. Scan the slide using GenePix scanner
4000B(Axon, U.S.A.) and analyze intensities of spots using
Nimblescan(Nimblegen, U.S.A.).
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