DNA for microarrays was prepared by amplification of cDNA cloned inserts using conventional PCR and the universal M13 forward (GTAAAACGACGGCCAGT) and reverse (CAGGAAACAGCTATGAC) vector primers (pGEM-Teasy), or M13 forward (GTAAAACGACG GCCAGT) and KS reverse (TCGAGGTCGACGGTATC) vector primers (pBII KS), with cDNA-containing plasmids as PCR templates. The microarray slides contained cDNA/EST clones described previously [1,2]. Control gene fragments cloned into the pBluescript vector (pBII KSt; Stratagene) were processed together with the cDNA/ESTs during microarray preparation. A complete set of 16 224 amplified cDNA inserts + control DNAs (672) was used to print a custom 16K BY-2 microarray (16,896). The complete microarray consisted of four microarray Bar Coded Slides, Type 7 star* or Lucidea (Amersham Biosciences UK Limited, Buckinghamshire, UK), with each PCRamplified cDNA sample spotted in duplicate on the slides. Standard slide arrangement consisted of 24 blocks with 12 rows and 32 columns each. The Array Spotter Generation III (Molecular Dynamics, Sunnyvale, CA, USA) was used for printing of slides. Code and description of control spots that are repeatedly spotted on each sub-array slide. Controls are spotted as bottom row in each individual block. Control spots 18-23 hybridize to Kanamycin positive mRNA spike from Promega that was added to each sample before cDNA synthesis. 1-NPTII,neomycin phosphotransferase II [V00618.1] 2-HYG,hygromycin B phosphotransferase [K01193.1] 3-RFP,RFP - red fluorescent protein [AF168419.2] 4-GUS,GUS - beta-glucuronidase protein [U12639] 5-PolyA,poly A oligonucleotide hybridization control [synthetic] 6-STS1,Yeast STS1 gene negative control [Z37996.1] 7-PBKSII,(M13-KS) vector pBluescript KSII (+) sequence [commercial] 8-pGEMTe,(M13-M13) vector pGEM-Teasy sequence (Self-ligated) [commercial] 9-Act200,200 bp PCR amplified fragment of tobacco actin gene [X63603] 10-Tub200,200 bp PCR amplified fragment of tobacco tubulin gene [NTU91562] 11-RibS4,PCR amplified fragment of tobacco ribosomal protein S4 gene [BP129322] 12-RibS29,PCR amplified fragment of tobacco ribosomal protein S29 gene [BP129273] 13-Actin,PCR amplified fragment of tobacco actin gene [BP534187] 14-Tubulin,PCR amplified fragment of tobacco tubulin gene [BP534511] 15-18SRNA,tobacco 18S rRNA gene [AJ236016] 16-SsDNA,Salmon sperm DNA (high complexity non-specific hybridization control) [commercial] 17-lambda,HindIII digested lambda phage DNA (low complexity non-specific hybridization control) [commercial] 18-Kan200,Promega kanamycin control RNA (Cat # C1381) corresponding DNA spot (spike control) [AY947641] 19-Kan200/2,Promega kanamycin control RNA corresponding DNA spot diluted 2x (spike control) [AY947641] 20-Kan200/5,Promega kanamycin control RNA corresponding DNA spot diluted 5x (spike control) [AY947641] 21-Kan200/10,Promega kanamycin control RNA corresponding DNA spot diluted 10x (spike control) [AY947641] 22-Kan200/50,Promega kanamycin control RNA corresponding DNA spot diluted 50x (spike control) [AY947641] 23-Kan200/100,Promega kanamycin control RNA corresponding DNA spot diluted 100x (spike control) [AY947641] 24-Water,deionized water control (negative control) 25-cDNA/2,PCR amplified cDNApool from BY-2 cells (diluted 5x) (positive control calibration) [custom] 26-cDNA/5,PCR amplified cDNApool from BY-2 cells (diluted 10x) (positive control calibration) [custom] 27-cDNA/10,PCR amplified cDNApool from BY-2 cells (diluted 20x) (positive control calibration) [custom] 28-cDNA/50,PCR amplified cDNApool from BY-2 cells (diluted 50x) (positive control calibration) [custom] 29-cDNA/100,PCR amplified cDNApool from BY-2 cells (diluted 100x) (positive control calibration) [custom] 30-cDNA/500,PCR amplified cDNApool from BY-2 cells (diluted 200x) (positive control calibration) [custom] 31-cDNA/1000,PCR amplified cDNApool from BY-2 cells (diluted 500x) (positive control calibration) [custom] 32-cDNA/5000,PCR amplified cDNApool from BY-2 cells (diluted 1000x) (positive control calibration) [custom] [1] Matsuoka, K., Demura, T., Galis, I., Horiguchi, T., Sasaki, M., Tashiro, G. and Fukuda, H. (2004) A comprehensive gene expression analysis toward the understanding of growth and differentiation of tobacco BY-2 cells. Plant Cell Physiol. 45: 1280-1289. [2] Galis, I., Simek, P., Narisawa, T., Sasaki, M., Horiguchi, T., Fukuda, H. and Matsuoka, K. (2006) A novel R2R3 MYB transcription factor NtMYBJS1 is a methyl jasmonate-dependent regulator of phenylpropanoid-conjugate biosynthesis in tobacco. Plant J. 46: 573-592.
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