核糖体蛋白基因芯片的制作和临床试用

Production and Clinical Test of Ribosomal Protein Gene Microarray

【目的】制作核糖体蛋白(ribosomal protein,RP)基因芯片,探讨该芯片应用方法。【方法】设计、合成58mer寡核苷酸探针,打印芯片。收集溃疡性结肠炎(ulcerative colitis,UC)患者和正常自愿者结肠黏膜,提取总RNA。逆转录法Cy5-dCTP标记试验样品,试验样品包括正常组4例、混合正常组1例、UC 8例、UC混合1例,Cy3-dCTP标记共同参照,1例正常样品和共同参照进行Cy5-dCTP、Cy3-dCTP荧光交换。试验样品和共同参照配对杂交、扫描,应用BRB-TOOL(3.90)进行数据分析。【结果】制作了包括77条RP基因,2条RP类似基因(RPL26-like1、RPL7-like1)低密度芯片。UC混合样品杂交的信号值与UC 1～8分别杂交信号值之均数最为接近,健康人混合样品杂交的信号值和H 1～4分别杂交信号值之均数没有相近之处,但更接近于单样品杂交的结果;混合样品所得到的差异基因数明显少于样品分别杂交所得到的差异基因数。2例荧光交换Ratio值高度相关。【结论】成功制作了RP基因芯片。样品混合会影响杂交数据的统计,同时,均一化能轻微减弱荧光染料引起数据的偏差。

Objective To make ribosomal protein （RP） gene micorarray and to explore its application. Methods We firstly designed and synthesized 58 mer nucleotide probe, and printed the gene array. Secondly, we collected the colonic mueosa of ulcerative colitis （UC） patients and that of healthy volunteers, and extracted the total RNA. The samples included 4 samples from individual healthy volunteers, one sample of mixed healthy volunteers, 8 samples from individual UC patients, and one sample of mixed UC patients. Cy5-dCTP was used for marking the samples, the common reference sample was marked with Cy3-dCTP by reverse transcription, and Cy5-dCTP and Cy3-dCTP were exchanged between the sample of mixed healthy volunteers and a common reference. Paired hybridization of the test samples and common references was carried out with probes in the gene array, and then gene arrays were scanned and the data were analyzed with BRB-Array Tools software package 3.90. Results RP gene arrays had been successfully produced, which included 77 RP genes, and 2 low-density RP-like genes （ RPL26-1ikel and RPL7-1ikel ）. The signal value of the mixed UC sample was similar to the mean signal value of UC sample 1-8. The signal value of mixed healthy volunteer sample differed from the mean signal value of healthy volunteer sample 1-4, but was similar to the signal value of the hybridized sample of single healthy volunteer. The number of differential genes from the hybridization of mixed UC sample or mixed healthy volunteer sample was obviously less than that from the hybridization of individual UC sample or healthy volunteer sample. The ratio value of two samples after fluorescent dye exchange was highly relevant. Conclusion RP gene arrays have been produced successfully. The mixing of the sample will affect the statistic results of hybridization data, and normalization can slightly reduce data bias induced by fluorochrome.