[Construction and primary application of oligonucleotide microarray specialized for pancreatic adenocarcinoma-associated genes].
OBJECTIVE
To investigate the construction of oligonucleotide microarray specialized for pancreatic adenocarcinoma-associated genes and its application.


METHODS
Pancreatic cancer related genes were purposely selected, and oligonucleotide microarray was prepared by spotting oligonucleotide probes onto glass slides coated with APS-PDC. Total RNA were extracted from frozen tissues with TRIzol method according to the manufacturer's protocol, and purified with QIAGEN RNeasy Kit. Labeled cDNA targets for hybridizations were synthesized by reverse transcription from control- and cancer-total RNA samples in the presence of Cy5-dCTP and Cy3-dCTP, respectively. The labeled probes were hybridized with oligonucleotide microarray for 16 h to 18 h. Hybridized microarray was scanned by Agilent laser scanner, and the acquired image was analyzed by Imagene3.0 software. The intensity ratio of Cy3 and Cy5 were calculated. To confirm the expression profiles of these genes, quantitative reverse transcription-PCR (Q RT-PCR) was carried out with CDC25B and TUSC3 genes. The product of PCR were quantitated by comparative Ct method.


RESULTS
The signal of microarray hybridization was clear, and the images had a lower background and higher signal-noise ratio. The signal of positive control spots were uniform, and spots of negative control and blank signal were fairly low. In comparison with normal pancreas, 24 differential expressed genes were identified, which included 17 up-regulated and 7 down-regulated genes. The results of Q RT-PCR demonstrated that the expression of CDC25B and TUSC3 in pancreatic cancer were increased and decreased respectively, which consistent with microarray hybridization.


CONCLUSIONS
The oligonucleotide microarray specialized for pancreatic cancer are desirable for its specialty, flexibility and sensitivity, which can simultaneously and parallelly detect multiple pancreatic cancer-associated genes. In contrast to normal pancreatic tissues, the genes expression profile are different significantly in pancreatic cancer.
Shi
Xin
X
Department of General Surgery, Zhongda Hospital, Southeast University, Nanjing 210009, China. shixined@hotmail.com
Wei
Wen-jun
WJ
Gao
Nai-rong
NR
Cheng
Zhang-jun
ZJ
Tang
Yong-hui
YH
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000368:Aged; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D015345:Oligonucleotide Probes