摘要
目的:制备胶原沉积抑制因子(decorin)cDNA探针并初步检测组织中decorin mRNA。方法:用聚合酶链反应(PCR)法将地高辛掺入decorin cDNA片段;琼脂糖凝胶电泳观察地高辛掺入对decorin cDNA片段分子量的影响;乙醇沉淀法纯化探针、紫外分光光度法测定探针含量;斑点杂交法检测探针的灵敏度和特异性;原位杂交法检测石蜡包埋组织中decorin mRNA。结果:成功获得了地高辛标记decorin cDNA探针,但掺入D IG后使得同一decorin片段分子量增大;一次100μl PCR可产生11μg标记探针;探针的灵敏度为19 pg;探针的特异性因待测分子中decorin片段的比例而不同,当以1 005 bp的decorin片段和pGEX-3X-decorin为待测基因时,特异性分别为2和20 pg;标记探针可显示不同组织中decorin mRNA。结论:用PCR法高效地制备了灵敏度和特异性都很高的地高辛标记decorin cDNA探针,并成功地检测了不同组织中decorin mRNA水平。
Objective：To prepare cDNA probe of decorin and try to localize decorin mRNA Methods：Digoxin was incorporated into decorin cDNA fragment by PCR. The effect of digoxin initially in tissues. incorporation on the molecular weight of decorin cDNA fragment was observed by agarose gel electrophoresis. The probe was purified by alcohol sedimentation method, and content of the probe was evaluated by ultraviolet spectrophotometry. Sensitivity and specificity of the probe was detected by dot hybridization. Decorin mRNA in tissues embedded in paraffine was detected by in situ hybridization. Results： Decorin cDNA probe marked with digoxin was got successfully, but incorporation of digoxin resulted in increased molecular weight of the same decorin fragment. Marked probe of 11 μg was made through one PCR of 100μl. Sensitivity of probe was 19 pg. Specificity of probe varied according to different proportion of decorin fragment in target DNA. When 1005 bp decorin fragment and pGEX - 3X - decorin were used as target DNA, specificity of marked probe was 2 pg and 20 pg respectively. Then, decorin mRNA in different tissues was localized. Conclusion： Decorin cDNA probe with high sensitivity and specificity and marked with digoxin by PCR method is prepared efficiently, and decorin mRNA levels in different tissues can be detected successfully.
出处
《西北国防医学杂志》
CAS
2006年第5期321-324,共4页
Medical Journal of National Defending Forces in Northwest China
基金
甘肃省自然科学基金资助项目(ZS001-A23-058-Y)
