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目的探讨胰腺癌相关基因寡核苷酸芯片的构建,初步验证其在检测胰腺癌基因表达谱方面的应用。方法有目的地筛选胰腺癌相关基因,采用合成后点样的方法,制成寡核苷酸基因芯片。TRIzol 法抽提组织总 RNA,在 cDNA 第一链合成过程中,通过反转录酶将 CyDye 标记核苷酸直接掺入到 cDNA 中制备荧光探针,其中用 Cy3-dCTP 标记胰腺癌组织,Cy5-dCTP 标记正常胰腺组织。将荧光标记探针与芯片杂交16～18 h。用 Agilent 扫描仪进行扫描,Imagene3.0软件进行图像分析,计算两种荧光 Cy3与 Cy5信号强度的比值。挑选差异基因 CDC25B 和 TUSC3进行荧光定量 PCR(sybrgreen 方法)验证,PCR 产物的定量方法采用比较 Ct 法。结果芯片杂交扫描图像信号清晰,具有较低的整体背景和较高的信噪比,各阳性质控点信号均匀一致,阴性质控点和空白点信号低。与正常组织相比,胰腺癌组织中差异表达基因24条,占全部基因的25.5%,其中上调基因17条(18.1%),下调基因7条(7.4%)。荧光定量 PCR 验证,CDC25B 和 TUSC3基因在胰腺癌中的表达趋势与芯片实验的结果一致。结论本研究制备的胰腺癌相关基因芯片可同时、并行检测多种胰腺癌相关基因的表达改变,具有一定的特异性和敏感性。胰腺癌组织与正常胰腺组织相比,基因表达谱具有明显差异。
Objective To investigate the construction of pancreatic cancer related gene oligonucleotide microarray and to preliminary verify its application in the detection of pancreatic cancer gene expression profile. Methods Screening pancreatic cancer-related genes in a targeted manner, using synthetic point-like method, made of oligonucleotide microarray. TRIzol extraction of tissue total RNA, first cDNA synthesis in the process, by reverse transcriptase CyDye labeled nucleotide directly into the cDNA preparation of fluorescent probes, which Cy3-dCTP labeled pancreatic cancer, Cy5 -dCTP labeled normal pancreatic tissue. Fluorescently labeled probe hybridized with the chip 16 ~ 18 h. Scanning with an Agilent scanner, Imagene3.0 software performed image analysis and calculated the ratio of the fluorescence intensity of Cy3 to Cy5 for both fluorophores. The differential genes CDC25B and TUSC3 were selected and validated by the fluorescence quantitative PCR (sybrgreen method). The quantitative PCR method was compared with the Ct method. Results The chip hybridization scanning image signal was clear, with a lower overall background and higher signal-to-noise ratio. The signal of each positive control point was uniform and the signals of negative control point and blank point were low. Compared with normal tissues, 24 differentially expressed genes in pancreatic cancer tissues accounted for 25.5% of the total, of which 17 genes were up-regulated (18.1%) and 7 genes were down-regulated (7.4%). Fluorescent quantitative PCR confirmed that the expression trend of CDC25B and TUSC3 in pancreatic cancer was in accordance with the result of chip experiment. Conclusion The pancreatic cancer related gene chip prepared in this study can detect the expression changes of many pancreatic cancer related genes simultaneously and in parallel, with a certain specificity and sensitivity. Pancreatic cancer tissue compared with normal pancreatic tissue, the gene expression profile was significantly different.