title: [Optimization of cDNA microarray fabrication].
abstract: To optimize and screen the most suitable target gene length,concentration and printing solution in cDNA microarray, housekeeping genes, such as beta actin and GAPDH, were selected as targets and hepatitis B virus gene as negative control. The RT-PCR primers that spanned at least one intron and whose products were at between 189 bp and 1078 bp were designed with primer premier 5.0, so did the hepatitis B virus gene PCR primer. After polymerase chain reaction, the products were purified with ethanol and dissolved in 3xSSC, 50% DMSO and 0.5 mol/L carbonate buffer(pH=9.0)respectively. The concentrations of target genes were adjusted at 0.5 microg/microL, 1.0 microg/microL and 1.5 microg/microL. The hybridization signals had a good specificity. No signal showed in either negative control (HBV) or blank control (printing solution only). There was no significant difference in target gene lengths. The P value of beta actin (189 bp,491 bp,974 bp) and GAPDH (227 bp,552 bp,1078 bp) was 0.378 and 0.866 respectively. There was no significant difference among concentrations(P=0.648),too. However, the higher the concentration was, the stronger the signals would be. Among the three kinds of printing solution, 50% DMSO was the best (P=0.0001), while the other two had no difference by multi-comparison (P=0.142). The target gene at length between 200 bp and 1000 bp has got the same hybridization signals.50% DMSO printing solution and the target gene concentration of 0.5 microg/microl are suitable for good hybridization.
authors.affiliation: Oncology Department, The First Affiliated Hospital, China Medical University, Shenyang 110001, China. huang_bj@163.com
authors.forename: Bao-Jun
authors.initials: BJ
authors.lastname: Huang
authors.forename: Yu-Jie
authors.initials: YJ
authors.lastname: Zhao
authors.forename: Hui-Mian
authors.initials: HM
authors.lastname: Xu
authors.forename: Qun
authors.initials: Q
authors.lastname: He
authors.forename: Yu-Kui
authors.initials: YK
authors.lastname: Zhang
authors.forename: Ying-Ying
authors.initials: YY
authors.lastname: Xu
authors.forename: Jia-Ming
authors.initials: JM
authors.lastname: Ma