以尼龙膜为载体的基因芯片探针及样品合理浓度筛选

Gene chip probe using nylon membrane as carrier and the screening of sample reasonable density

背景:目前多数芯片采用玻璃片基,以Cy3和Cy5标记,价格高,主要用于科研,但不能在临床上大规模推广应用。课题组采用尼龙膜为载体制作基因芯片,以期获得一种具有高灵敏度和准确性、快速简便、可同时检测多种疾病且成本低的芯片。目的:对以尼龙膜为载体基因芯片的可行性进行检测,同时对所用探针及目的基因的量进行优化。设计、时间及地点:基因工程探针设计,于2005-10/2006-09在兰大科技园百源基因公司实验室完成。材料:甘肃省肿瘤医院2005-10/2006-04切除的新鲜大肠癌标本,取距肿瘤边缘5～10cm以上的正常组织作为对照,病理证实无癌细胞浸润。尼龙膜带正电荷,购自Amersco公司。方法:从液氮中取出冻存的新鲜组织200mg,采用Trizol法提取组织总RNA,并用Oligo dT纤维素层析法纯化mRNA。使用TaKaRa的M-MLV Rtase cDNA synthesis Kit(code D6130)反转录合成cDNA的第1,2链,产物分为两部分,一部分作为模板进行地高辛标记PCR,所得产物作为目的基因,另一部分作为制备探针的模板。对获得的GAPDH,Actin,Cyclin D1三种基因片段PCR产物进行纯化。以带正电荷的尼龙膜为载体,将不同浓度的三种基因片段作为探针点于其上,所得芯片分别与不同浓度的地高辛标记目的基因进行杂交。结果:目的基因取2.5μL,1.25μL时,加入杂交液后因目的基因的浓度太小,造成各点探针密度相对较高,而无法反映出正确的显色趋势。目的基因取5μL时,探针密度为10～20μL情况下3种探针显色深度较恰当的反映了由强到弱的趋势,ACTIN>CyclinD1>GAPDH。目的基因取10μL时,探针取15μL,20μL的量能较为合理的反映由强到弱的趋势。目的基因取20μL时,此时杂交液中目的基因的浓度对于ACTIN和CyclinD1过高,不能明显反映二者着色深浅变化。结论:以尼龙膜为载体的基因芯片本底清晰,合理目的基因用量为5μL,与其相匹配的优化探针用量为10～20μL。

BACKGROUND： At present most chips use the glass film base by Cy3 and Cy5 mark, their price is so high that are only applied in scientific researches, and clinical application cannot be promoted massively. The topic-based group uses the nylon membrane as the carder to manufacture gene chips, in order to obtain one kind of chip that has high sensitivity and accuracy, fast simple, may simultaneously examine many kinds of illnesses to get sick, and costs low. OBJECTIVE： To investigate the feasibility of taking the nylon membrane as the carder to obtain gene chip, and simultaneously optimize the quantity of the probe and the goal gene. DESIGN, TIME AND SETTING： Experiments at genetic engineering probe design were carried out from October 2005 to September 2006 in the laboratory of Lanzhou University Science and Technology Garden Baiyuan Gene Company. MATERIALS： Fresh colon cancer samples were excised from Gansu Cancer Hospital between October 2005 and April 2006, while the normal tissues apart from tumor edge above 5-10 cm were taken as controls. The pathology confirmed that the cancer cell had not infiltrated. The nylon membrane belt （from Amersco Corporation） was positive charge. METHODS： 200 mg cryopreserved tissues were taken out of liquid nitrogen, total RNA extraction was performed using Trizol method, and mRNA was purified through the Oligo dT chromatography. The first and the second chain of cDNA was synthesized with reverses transcription by using TaKaRa＇s M-MLV Rtase cDNA synthesis Kit （code D6130）. The product was divided into two parts, a part as the template for the digoxin mark PCR, the obtained product served as the goal gene. Another part was taken as the template to manufacture probe. As for the resultant GAPDH, Actin, CyclinD1, the gene fragment PCR product was processed into the purification. Using the positively charged nylon membrane as the carrier,three kind of gene fragment at different density were taken as probes on the carrier, the obtained chip hybridizated separately with the different densities of digoxin mark goal gene. RESULTS： When the goal gene （2.5 μ L and 1.25 μ L） was sampled and added with the hybrid fluid, the goal gene＇s density was so small as to increase the density on each probe, which was unable to reflect the correct colored tendency. When the goal gene was sampled 5 μ L and the probe density was 10-20 μ L, three kinds of probes exhibited a colored depth tendency arranging from strong to weak, ACTIN〉CyclinD1〉GAPDH. When the goal gene was sampled 10 μ L, the probe density was 15 μ L, 20 μ L was a reasonable dose to reflect a strong-weak tendency. When the goal gene was sampled 20 μ L, the density of goal gene in the hybrid fluid was excessively high regarding ACTIN and CyclinD1, which was unable to reflect the changes of coloration depth. CONCLUSION： Using the nylon membrane as the carder to produce gene chip, the chip background is clear. The reasonable amount of goal gene used is 5 μ L, with the optimized probe amount which it matches is 10-20 μ L.