Inoue Kaoru, Sakurada Yosuke, Murakami Masaru, Shirota Mariko, Shirota Kinji
Research Institute of Biosciences, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa, 229-8501, Japan.
Virchows Arch. 2003 Feb;442(2):159-62. doi: 10.1007/s00428-002-0719-5. Epub 2002 Oct 29.
The recent development of the laser microdissection (LMD) technique enables one to target particular tissues or cells for gene or protein analyses. The purpose of this study was to detect local mRNA expression of vascular endothelial growth factor (VEGF) and its receptor, flk-1, in the glomeruli of normal rat kidneys using the LMD system. Frozen sections of the kidney of 8-week-old male Wistar rats were made. The glomeruli were dissected from the frozen sections with the LMD system, and total RNA was extracted from 200 glomeruli in each kidney. Reverse-transcription polymerase chain reaction (RT-PCR) revealed the local mRNA expression of three isoforms of VEGF, flk-1 and GAPDH in the glomeruli. Moreover, the real-time PCR was performed to evaluate the experimental condition for quantification of VEGF and flk-1 mRNA expression using this system, and the results showed that at least 10 glomeruli might be needed for quantifying local VEGF mRNA expression. However, cDNA from 200 glomeruli was not enough for quantitative evaluation of flk-1 mRNA with this system. These results demonstrate the reproducibility of the analysis of mRNA expression in the renal glomeruli using the LMD system and also suggest that the application of the LMD technique will provide information to further our understanding of the mechanisms involved in kidney diseases.
激光显微切割（LMD）技术的最新进展使人们能够针对特定组织或细胞进行基因或蛋白质分析。本研究的目的是使用LMD系统检测正常大鼠肾脏肾小球中血管内皮生长因子（VEGF）及其受体flk-1的局部mRNA表达。制备了8周龄雄性Wistar大鼠肾脏的冰冻切片。用LMD系统从冰冻切片中分离出肾小球，并从每个肾脏的200个肾小球中提取总RNA。逆转录聚合酶链反应（RT-PCR）显示了肾小球中VEGF的三种异构体、flk-1和甘油醛-3-磷酸脱氢酶（GAPDH）的局部mRNA表达。此外，进行实时PCR以评估使用该系统定量VEGF和flk-1 mRNA表达的实验条件，结果表明，定量局部VEGF mRNA表达可能至少需要10个肾小球。然而，来自200个肾小球的cDNA不足以用该系统对flk-1 mRNA进行定量评估。这些结果证明了使用LMD系统分析肾小球中mRNA表达的可重复性，也表明LMD技术的应用将为进一步了解肾脏疾病的发病机制提供信息。
