title: 32K Cotton cDNA array
status: Public on Jan 01 2008
contact.city: Beijing
contact.first: Yu-Xian
contact.last: Zhu
contact.email: zhuyx@water.pku.edu.cn
contact.address: 5# Summer Palace Road
contact.country: China
contact.institute: College of life sciences, Peking University
contact.phone: 86-10-62751193
organism: Gossypium hirsutum
manufacture_protocol: cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine's linear RNA amplification method and subsequent enzymatic reaction. In detail, double-stranded cDNAs (containing the T7 RNA polymerase promoter sequence) were synthesized from 10 ¦Ìg total RNA using the cDNA synthesis system according to the manufacturer's protocol (TaKaRa). A T7-oligo(dT) primer (5'-AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCTTTTTTTTTTTTTTTTT-3') was used instead of the poly-T primer provided in the kit. After completion of double-stranded cDNA synthesis, cDNA products were purified using a PCR purification kit (Qiagen) and eluted with 60 ¦ÌL elution buffer. One-half of the eluted double-stranded cDNA products were vacuum evaporated to 8 ¦ÌL and subjected to 20-¦ÌL in vitro transcription reactions at 37¡ãC for 3 h using the T7 RiboMAX Express large scale RNA production system (Promega). The amplified RNA was purified using the RNeasy mini kit (Qiagen). Klenow enzyme labeling strategy was adopted after reverse transcription. Briefly, 2 ¦Ìg amplified RNA was mixed with 2 ¦Ìg random hexamers, denatured at 70¡ãC for 5 min, and cooled on ice. Then, 4 ¦ÌL of first-strand buffer, 2 ¦ÌL of 0.1 M DTT, 1 ¦ÌL 10 mM dNTP, and 1.5 ¦ÌL SuperScript II (Invitrogen) were added. The mixtures were incubated at 25¡ãC for 10 min, then at 42¡ãC for 60 min. The cDNA products were purified using a PCR purification kit (Qiagen) and vacuum evaporated to 10 ¦ÌL. The cDNA was mixed with 2 ¦Ìg random nanomer, heated to 95¡ãC for 3 min, and snap cooled on ice. Then, 10 ¦ÌL buffer, dNTP, and Cy5-dCTP or Cy3-dCTP (Amersham Pharmacia Biotech) were added to final concentrations of 120 ¦ÌM dATP, 120 ¦ÌM dGTP, 120 ¦ÌM dTTP, 60 ¦ÌM dCTP, and 40 ¦ÌM Cy-dye. Klenow enzyme (1 ¦ÌL; TaKaRa) was then added, and the reaction was performed at 37¡ãC for 60 min. Labeled cDNA was purified with a PCR purification kit (Qiagen) and resuspended in elution buffer. Labeled controls and test samples were quantitatively adjusted based on the efficiency of Cy-dye incorporation and mixed with 30 ¦ÌL hybridization solution (50% formamide, 1x hybridization buffer; Amersham Biosciences). DNA in hybridization solution was denatured at 95¡ãC for 3 min prior to loading onto a microarray. Arrays were hybridized at 42¡ãC overnight and washed with two consecutive solutions (0.2% SDS, 2x SSC at 42¡ãC for 5 min, and 0.2x SSC for 5 min) at room temperature.