title: Narusaka Lab. Arabidopsis 1.2K Full-length cDNA microarray ver.1
status: Public on Feb 08 2007
contact.city: Kaga-gun, Okayama
contact.first: Yoshihiro
contact.last: Narusaka
contact.email: yo_narusaka@bio-ribs.com
contact.address: 7549-1, Kibityuo-town Yoshikawa
contact.country: Japan
contact.institute: Research Institute for Biological Sciences
contact.phone: 81-866-56-9450
organism: Arabidopsis thaliana
description: A total of 1.2K potential stress-related genes that encode known or putative protein were selected for this study from the genes covered by Arabidopsis 7K array (RIKEN, Japan) and Arabidopsis oligo microarray (Agilent technologies, USA), which these genes include biotic and abiotic stress-inducible genes (Seki M et al. Plant J 31: 279-292, 2002; Narusaka Y et al. Plant Cell Physiol 44: 1246-1252, 2003; Narusaka Y et al. Plant Cell Physiol 44: 377-387, 2003; Narusaka Y et al. Mol Plant-Microbe Interact 17: 749-762, 2004)
  
  
manufacture_protocol: In the cDNA microarray analyses, we used ca.1.2K cDNA clones and the lambda control template DNA fragment (TX803; Takara) as an external control. The vectors used for cDNA library construction were modified lambda ZAP (Carninci et al. (1996) Genomics, 37, 327-336) and lambda FLC-1(Carninci et al. (2001) Genomics, 77, 79-90). Inserts of cDNA clones were amplified by PCR using primers complementary to vector sequences flanking both sides of the cDNA insert, as described previously (Seki et al. (2001) Plant Cell, 13, 61-72). PCR products were precipitated in isopropanol and the DNA was resuspended in 1 x Micro Spotting Solution (Arrayit; Telechem International Inc., Sunnyvale, CA). One aliquot of each finished reaction was electrophoresed on a 1 % agarose gel to confirm amplification quality and quantity. PCR products were arrayed from 384-well microtitre plates onto a micro slide glass (model Super Aldehyde substrates; Telechem International Inc., Sunnyvale, CA) using the microarray stamping machine (model SpotArray24; PerkinElmer, MA, USA).
  
  Hybridization procedures and parameters (The protocol and conditions used during
  hybridization, blocking and washing):
  The slides were postprocessed according to the manufacturer's protocol (Telechem International Inc.). The printed slides were dried (RH < 30 %) and subjected to UV cross-linking. They were rocked in 0.2 % SDS for 2 min three times and then rocked in distilled water for 2 min twice vigorously. The slide racks were transferred into a chamber containing boiling water and left for 2 min. The blocking solution containing 1 g sodium borohydride, 300 ml PBS and 90 ml 100 % ethanol was poured into the glass chamber. The slide racks were shaken gently for 5 min, then transferred into a chamber containing 0.2 % SDS and shaken gently for 1 min three times. They were transferred into a chamber containing distilled water, shaken gently for 1 min, and dried by centrifugation for 20 min.The probe samples prepared as
  described previously were denatured by placing them in a 100C heat block for 2
  min, left at room temperature for 5 min, and then used for hybridization. The slides
  were placed in a sealed hybridization cassette (Telechem International Inc.,
  Sunnyvale, CA) and submerged in a 65C water bath for 16 to 20 h. After
  hybridization, slides were washed in 2 X SSC, 0.03% SDS for 2 min, then in 1 X
  SSC for 2 min, and finally in 0.06 X SSC for 2 min. Then, the slides were
  immediately dried by centrifugation (2 min at 180 g). Slides were scanned with a
  ScanArray (PerkinElmer, Wellesley, MA, USA) as described previously (Seki et al.
  (2001) Plant Cell 13:61-72).