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Platform GPL2610 Query DataSets for GPL2610
Status Public on Jan 01, 2006
Title Upland Cotton Fiber-Specific cDNA Array
Technology type spotted DNA/cDNA
Distribution custom-commercial
Organism Gossypium hirsutum
Manufacturer CapitalBio Corporation, National Engineering Research Center for Beijing Biochip Technology, 18 Life Science Parkway, Beijing 102206, China
Manufacture protocol 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.
Description 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.
Contributor(s) Shi Y, Zhang L, Cheng J, Zhu Y
Citation(s) 16461577
Submission date Jul 08, 2005
Last update date Sep 08, 2006
Contact name Yu-Xian Zhu
Phone 86-10-62751193
Fax 86-10-62754427
Organization name College of life sciences, Peking University
Lab National Lab of Protein Engineering & Plant Genetic Engineering
Street address 5# Summer Palace Road
City Beijing
ZIP/Postal code 100871
Country China
Samples (66) GSM63341, GSM63342, GSM63343, GSM63344, GSM63345, GSM63346 
Series (2)
GSE2901 cDNA array expression profile of wild-type and fl-mutant upland cotton ovules
GSE18028 cDNA microarray expression profile during fiber elongation between G. hirsutum and G. barbadense

Data table header descriptions
GB_ACC Accession numbers of ESTs used for the array
Gene info information about the gene represented by the sequence in the spot

Data table
CM001A01 DR452281 calnexin - soybean
CM001A02 DR452282 F-box and Tub-domain-containing protein [Arabidopsis thaliana]
CM001A03 DR452283
CM001A04 DR452284 nectarin 5 [Nicotiana langsdorffii x Nicotiana sanderae]
CM001A05 DR452285 ATP-dependent clp protease ATP-binding subunit clpA homolog, chloroplast precursor
CM001A06 DR452286
CM001A07 DR452287 expressed protein [Arabidopsis thaliana]
CM001A08 DR452288 late embryogenesis-abundant protein Lea5-A - upland cotton
CM001A09 DR452289 protein kinase homolog M3E9.30 - Arabidopsis thaliana
CM001A10 DR452290 probable ATP synthase chain - soybean
CM001A11 DR452291
CM001A12 DR452292 ABA-responsive element binding protein 3 [Arabidopsis thaliana]
CM001B01 DR452293
CM001B02 DR452294 homeodomain leucine zipper protein [Oryza sativa]
CM001B03 DR452295 hypothetical protein XP_149431 [Mus musculus]
CM001B04 DR452296 hypothetical protein T1K7.8 [imported] - Arabidopsis thaliana
CM001B05 DR452297 WD-40 repeat protein family [Arabidopsis thaliana]
CM001B06 DR452298 putative reverse transcriptase [Oryza sativa]
CM001B07 DR452299 fiber protein Fb10 [Gossypium barbadense]
CM001B08 DR452300

Total number of rows: 11706

Table truncated, full table size 1517 Kbytes.

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp

Supplementary data files not provided

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