中文摘要：

目的探索建立稳定可靠的单细胞RT-PCR实验体系。方法以人β-actin和DPH2L基因为靶基因，通过各实验条件的比较优化，对单细胞RT-PCR扩增单个细胞中特异性目的基因和全细胞mRNA两种方法的扩增效果进行深入研究，并结合琼脂糖凝胶电泳和DNA测序技术，分析该方法的灵敏性、特异性、重复性和可靠性。结果单个细胞中的全部mRNA均得到了扩增，扩增成功率为75％，产物经核酸定量均可达到懈水平，足够常规分子生物学实验之用。β-actin和DPH2L基因均得到特异性扩增产物，未见基因组DNA污染，经BLAST比对为100％吻合，不同条件下扩增成功率在30％-90％之间。结论单细胞RT-PCR是一种行之有效的单细胞基因表达的研究工具，提高Mg2^＋反应浓度并采用巢式引物有利于提高扩增成功率。

英文摘要：

Objective To seek and establish a stable reliable single cell RT-PCR technique. Methods Whole cell mRNAs or human β-actin and DPH2L gene were amplified by single cell RT-PCR respectively. During this process different experimental conditions were optimized and amplification effects of special target gene and the whole mRNAs were studied. The sensitivity efficiency and fidelity of single cell RT-PCR were investigated by agarose gel electrophoresis and DNA sequencing. Results The whole mRNAs in single cell were amplified effectively（75% ） by single cell RT-PCR, the quality of which was up to lag level. Human β-actin and DPH2L gene were amplified successfully by single cell RT-PCR and the sequence of PCR products was completely matched with Genebank. The percentage of successful reactions was from 30% to 90% under different experimental conditions. Conclusion Single cell RT-PCR is indeed a powerful tool to study gene expression on single cell level. High magnesium ion concentration and nest primers contributed to amplify successfully.