CapitalBio Corporation, National Engineering Research Center for Beijing Biochip Technology, 18 Life Science Parkway, Beijing 102206, China
Individual bacterial clones were selected and distributed into 96-well plates. PCR amplification of DNA using primers specific to the plasmid vector sequences flanking the insert cDNA (M13 forward and reverse primers) were performed in 96-well PCR plate in a Perkin-Elmer 9600 thermocycler in 50-µL reactions containing 1× PCR buffer (TaKaRa, Dalian, China), 2.0 mM MgCl2, 0.2mM dNTPs, 10 pmol of each primer, 5 units of Taq polymerase, and 10 ng plasmid template. The PCR cycle consisted of one round at 95℃ for 3 min, then 35 cycles including 95℃ for 1 min, 55℃ for 20 s and 72℃ for 90 s, with a final extension at 72℃ for 5 min. One µL of the PCR reactions was analyzed in a 1% agarose gel to verify the success of the amplification. The remaining PCR products were precipitated with addition of 100 µL of anhydrous ethanol and resuspended in 15 µL of 50% DMSO for arraying. As a result, 11,692 cDNA were amplified and printed on the cDNA microarrays. Fluorescent dye (Cy5 and Cy3-dCTP) labeled DNA was produced through Eberwine's linear RNA amplification method and subsequent enzymatic reaction. Double-stranded (ds) cDNA containing T7 RNA polymerase promoter sequence was synthesized from 10 µg of total RNA using a cDNA synthesis kit following the manufacturer’s instructions (TaKaRa). A T7-OligodT primer (5’-AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCTT TTTTTTTTTTTTTTTV -3’) was used to replace the poly T primer provided in the kit. Synthesized ds cDNA was purified using the PCR Purification kit (Qiagen) and purified products were eluted using 60-µL elution buffer. A half of the eluted ds cDNA was reduced to 8 µL before subjecting to in vitro transcription in 20 µL of reaction mixture using the T7 RiboMAX Express Large Scale RNA Production System (Promega) for 3 h at 37℃. Amplified RNA (aRNA) was purified using the RNeasy Mini kit (Qiagen). For labeling of the probes, 2 µg of aRNA was mixed with 2 µg of random hexamers, denatured at 70℃ for 5 min, cooled on ice before adding 4 µL of first strand synthesis buffer, 2 µL of 0.1M DTT, 1 µL 10 mM mixed dNTPs, and 1.5 µL SuperScript II (Invitrogen) to the system. The reaction mixture was first incubated at 25℃ for 10 min before transferring to 42℃ for 60 min. The labeled cDNA was purified using a PCR Purification kit (Qiagen), reduced to 10 µL volume and mixed with 2 µg of random nonamers, heated to 95°C for 3 min and cooled on ice immediately. 10×buffer, dNTP and Cy5- or Cy3-dCTP (Amersham Pharmacia Biotech) was added to a final concentration of 120 µM each for dATP, dGTP and dTTP, 60 µM for dCTP and 40 µM for Cy5 or Cy3 dyes respectively. One µL Klenow (Takara) was added to the mixture and the reaction progressed for 60 min at 37℃. The labeled cDNA was again purified with a PCR Purification kit (Qiagen), resuspended in elution buffer and quantitatively adjusted based on the efficiency of dye incorporation. Appropriate amount of the labeled cDNA was mixed in a final volume of 30 µL of hybridization solution that contains 50% formamide and 1×hybridization buffer (Amersham Bioscience), and was denatured at 95℃ for 3 min prior to loading onto a microarray. The array was hybridized at 42℃ overnight and was first washed for 5 min at 42°C with washing solutions that contain 0.2% SDS and 2×SSC followed by a second wash using washing solutions that contain 0.2% SSC for 5 min at room temperature.
To estabilish upland cotton fiber-specific cDNA array, we constructed cDNA library from 5-10 day post anthesis cotton fibers when fiber cells elongate most quickly. Secondly, we randomly sequenced over 35,000 ESTs from this library and acquired a gene pool of more than 11,000 UniESTs. The cotton UniESTs were then PCR-amplified and printed onto microarray. This array is comprised of 11,692 high-quality cotton cDNAs (each sequence length>300bp, average length = 586bp) representing over 8,000 cotton genes.