AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed. To better understand cellular functions, the isolation of single cells and subsequen...AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed. To better understand cellular functions, the isolation of single cells and subsequent quantification of the expressed genes is essential. METHODS: Normal liver tissue was obtained from operation, snap-frozen in liquid nitrogen and sectioned in crystat. Individual hepatocytes were microdissected. RNA was extracted, then reverse transcribed and amplified using real-time quantitative polymerase chain reaction (PCR). RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide. In this way, cells were collected, RNA was extracted, reverse transcribed to cDNA and used for analysis of RNA expression by real-time quantitative PCR. The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells. CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real-time PCR. These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.展开更多
文摘AIM: The manner in which a cell responds to and influences its environment is ultimately determined by the genes that are expressed. To better understand cellular functions, the isolation of single cells and subsequent quantification of the expressed genes is essential. METHODS: Normal liver tissue was obtained from operation, snap-frozen in liquid nitrogen and sectioned in crystat. Individual hepatocytes were microdissected. RNA was extracted, then reverse transcribed and amplified using real-time quantitative polymerase chain reaction (PCR). RESULTS: Single hepatocytes were dissected by laser beam and catapulted to the microcentrifuge cap which was put above the slide. In this way, cells were collected, RNA was extracted, reverse transcribed to cDNA and used for analysis of RNA expression by real-time quantitative PCR. The amplification results showed that quantitation of the RNA inside the cell was compatible with the number of cells. CONCLUSION: The expression of RNA in single cells can be quantitated successfully by using laser microdissection and real-time PCR. These techniques provide an opportunity to monitor in vivo gene expression levels in single hepatocytes.
