应用激光显微切割技术检测单个结肠细胞的基因表达

LASER MICRODISSECTION OF A SINGLE CELL FROM COLON TISSUE FOR GENE ANALYSIS

目的 :探讨检测单个结肠细胞的基因表达的方法。方法 :应用激光显微切割技术 (lasermicrodissection)从冰冻切片上将单个结肠细胞切下 ,提取总RNA ,将RNA逆转录成cDNA ,采用巢式逆转录聚合酶链反应 (nestedRT PCR)检测mRNA的表达。结果 :在显微镜下用紫外激光显微切割机 ,将单个结肠细胞成功切下 ,提取RNA后 ,逆转录成cDNA ,经过巢式RT PCR扩增后 ,扩增产物在琼脂糖凝胶上清晰可见。结论 :联合应用激光显微切割和巢式RT

Aim: To investigate the method of detecting gene expression in colon tissue at a single cell level. Methods: Individual cell(s) were picked up from colon frozen section using laser microdissection. RNA was extracted, reverse transcripted to complementary DNA (cDNA). cDNA was then analyzed by nested reverse transcription polymerase chain reaction (nested RT PCR) using two pairs of primers. Results: Single cell(s) were selectively picked up using an ultraviolet laser micromanipulator. RNA was extracted, reverse transcripted and used for nested RT PCR. Amplification products of cDNA from down to a single cell could be clearly visualized in the agarose gel. Conclusion: The combined utilization of laser microdissection and nested RT PCR provides an opportunity to analyze gene expression at single cell(s) level in colon tissue.