胰腺癌相关基因寡核苷酸芯片的制备和初步应用(英文)

Construction and preliminary application of oligonucleotide microarray specialized for pancreatic adenocarcinoma

目的:研究胰腺癌相关基因寡核苷酸芯片的构建及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将合成的寡核苷酸探针点于同型双功能偶联剂(APS-PDC)包被的基片上,制成寡核苷酸基因芯片。Trizol法抽提组织总RNA,并用Qiagen Rneasy kit进一步纯化。在cDNA第1链合成过程中,通过反转录酶将CyDye标记核苷酸直接渗入到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。将荧光标记探针定量后与芯片杂交16-18 h。用Agilent扫描仪进行扫描,Imagene3.0软件(Biodiscovery,Inc.)图像分析,计算2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PUR(Sybrgreen方法)验证,以B-actin基因为内参照基因,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(占18.1%),下调基因7条(占7.4%)。荧光定量PCR验证,CDC25B和TUSC3基因在胰腺癌中的表达分别为上调和下调,其表达趋势与芯片实验的结果一致。结论: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灵敏性。胰腺癌组织与正常胰腺组织相比,基因表达谱具有明显的差异,为进一步研究胰腺癌的发病机理和早期诊断提供了有用的信息。

AIM ： To investigate the construction of oligonucleotide microarray specialized for pancreatic adenocarcinoma - associated genes and its preliminary application of detecting differential expressed genes in pancreatic cancer. METHODS： Pancreatic cancer related genes were purposely selected, and oligonucleotide microarray was prepared by spotting oligonucleotide probes on glass slides coated with APS - PDC. Labeled cDNA targets for hybridizations were synthesized by reverse transcription from total RNA in the presence of Cy5 - dCTP and Cy3 - dCTP, respectively. Hybridized microarray was scanned by Agilent laser scanner, and the aquired image was analyzed by Imagene3.0 software. The intensity ratios of Cy3 and Cy5 were calculated. To confirm the expression profiles of these genes, quantitative reverse transcription - PCR （ QRT - PCR） was carried out for CDC25B and TUSC3 genes, and β - actin gene was taken as internal control. The product of PCR was 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. In comparison with normal pancreas, twenty - four differentially expressed genes were identified which included seventeen up - regulated and seven down - regulated genes. The results of QRT - PCR demonstrated that the expressions of CDC25B and TUSC3 in pancreatic cancer were up - regulated and down - regulated respectively, which is consistent with microarray results. CONCLUSION ： The oligonucleotide microarray specialized for pancreatic cancer is desirable for its specialty and sensitivity, which can simultaneously and parallelly detect multiple pancreatic cancerassociated genes.