Shi Xin, Wei Wen-jun, Gao Nai-rong, Cheng Zhang-jun, Tang Yong-hui
Department of General Surgery, Zhongda Hospital, Southeast University, Nanjing 210009, China.
Zhonghua Wai Ke Za Zhi. 2007 Jan 1;45(1):39-42.
To investigate the construction of oligonucleotide microarray specialized for pancreatic adenocarcinoma-associated genes and its application.
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.
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.
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.
研究胰腺癌相关基因专用寡核苷酸微阵列的构建及其应用。
特意选取胰腺癌相关基因，通过将寡核苷酸探针点样到涂有APS - PDC的载玻片上制备寡核苷酸微阵列。按照制造商的方案，采用TRIzol法从冷冻组织中提取总RNA，并用QIAGEN RNeasy试剂盒进行纯化。分别在Cy5 - dCTP和Cy3 - dCTP存在的情况下，从对照和癌组织总RNA样本通过逆转录合成用于杂交的标记cDNA靶标。将标记的探针与寡核苷酸微阵列杂交16至18小时。用安捷伦激光扫描仪扫描杂交后的微阵列，并用Imagene3.0软件分析获取的图像。计算Cy3和Cy5的强度比。为确认这些基因的表达谱，对CDC25B和TUSC3基因进行定量逆转录 - PCR（Q RT - PCR）。通过比较Ct法对PCR产物进行定量。
微阵列杂交信号清晰，图像背景较低，信噪比高。阳性对照点的信号均匀，阴性对照和空白信号点相当低。与正常胰腺相比，鉴定出24个差异表达基因，其中包括17个上调基因和7个下调基因。Q RT - PCR结果表明，CDC25B和TUSC3在胰腺癌中的表达分别升高和降低，这与微阵列杂交结果一致。
胰腺癌专用寡核苷酸微阵列因其特异性、灵活性和敏感性而理想，可同时并行检测多个胰腺癌相关基因。与正常胰腺组织相比，胰腺癌中的基因表达谱有显著差异。
