A normal neonatal human diploid fibroblast strain MJ90 was cultured in Dulbecco's modified Eagle's minimal essential medium (DMEM; Sigma-Aldrich Japan) supplemented with 2 mM L-glutamine (Life Technologies) and 10% fetal calf serum (FCS; Life Technologies). A normal human umbilical vein endothelial cell strain HUE142-2 were grown in MCDB151 medium (Sigma-Aldrich Japan) supplemented with 15% fetal bovine serum, 5 µg/ml of heparin (Sigma), and 5 ng/ml of recombinant acidic fibroblast growth factor in a plastic flask (Corning) precoated with bovine fibronectin solution (1 µg/cm2, Wako).The 40 µg of plasmid hTRT/FLAG or hTERTn2 (from Prof. F. Ishikawa, Kyoto University)linealized by a single cut with a restriction enzyme NruI was transfected into 10^7 MJ90 cells at PDL 41 or the HUE142-2 cells at PDL 35 by electroporation (Invitrogen) at 330 V. Clones harboring the plasmids were obtained by 400 µg/ml of G418 selection (Wako) and named as TF1 from MJ90 and nTE4-5 from the HUE142-2 cells. The TF1 cells at PDL 104, 148, and 216 and nTE4-5 cells at PDL 105, 145, and 216 were collected and stored at –80°C until use. Total RNA was extracted from the cultured cells using an RNAeasy™ total RNA isolation kit (Qiagen) according to the protocols the manufacturer recommended. The poly(A)-RNA was isolated from the extracted total RNA using µMACS™ mRNA isolation kit(Miltenyi Biotec).One-microgram Poly(A)-RNA samples extracted from cell strains after hTERT induction were labeled with the fluorescent dye Cy-3 by random-primed reverse transcription. Similarly, 1-µg Poly(A)-RNA samples extracted from MJ90 cells at PDL 30 and the HUE 142-2 cells at PDL 38 before hTERT induction were labeled with Cy-5, and used as the expression references. Each pair of Cy-3 and cy-5 labeled cDNAs were hybridized as probes on RIKEN human 21K arrays. The array contained 21,168 spots in 48 blocks (4 x 12), each containing 441 spots (21 x 21). Among them, 20,784 spots are human cDNA clones purchased from ResGen™ (Invitrogen Corporation) and the remaining spots are the internal and exogenous controls spotted on every subgrid as positive and negative controls. All microarray analyses on the six pairs, i.e. MJ90 vs. TF1-PDL104, TF1-PDL148, or TF1-PDL216 and HUE142-2 vs. nTE4-5-PDL105, nTE4-5-PDL145, or nTE4-5 -PDL216 were carried out in duplicate. (_1 & _2). Arrays were laser-scanned using ScanArray 5000 confocal laser scanner (GSI Lumonics), and the fluorescence measurements were made separately by channels 1 and 2 for each dye at each spot on the array. Let CH1I and CH2I be spot intensities scanned by channels 1 and 2, respectively. Let Ch1B and CH2B be their background intensities. In this study, the values of CH1 = CH1I-CH1B and CH2 = CH2I – CH2B were used as gene expression levels in each sample. Each result of the duplicated microarray experiment was independently subjected to global normalization and new normalization (Ohtaki et al., Technical Report Series of Statistical Research Group, Hiroshima Univershity, No.04-07).
Keywords: time-course
Less...