Yong Voon Chen, Ong Khang Wei, Sidik Shiran Mohd, Rosli Rozita, Chong Pei Pei
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra Malaysia, Serdang, Selangor, Malaysia.
J Microbiol Methods. 2009 Nov;79(2):242-5. doi: 10.1016/j.mimet.2009.08.019. Epub 2009 Sep 6.
In situ Reverse Transcriptase PCR (in situ RT-PCR) can amplify mRNA and localize gene expression in cells. However, this method is not feasible in fungi as the thick fungal cell wall constitutes a barrier to this procedure. We developed a two step in situ RT-PCR procedure which enabled the detection and localization of Candida tropicalis mRNA expression in formalin-fixed, paraffin-embedded (FFPE) mouse kidney sections. This in situ hybridization study revealed the first direct evidence for deposition of Candida tropicalis secreted aspartic proteinase 2 (CtSAP2) in the tip of pseudohyphae and its involvement in acute systemic candidiasis. We conclude that in situ RT-PCR can be successfully applied to FFPE tissues and will offer new perspectives in studying gene expression in Candida species.
原位逆转录聚合酶链反应（原位RT-PCR）可扩增信使核糖核酸（mRNA）并定位细胞中的基因表达。然而，该方法在真菌中不可行，因为厚厚的真菌细胞壁对该操作构成了障碍。我们开发了一种两步原位RT-PCR程序，能够在福尔马林固定、石蜡包埋（FFPE）的小鼠肾脏切片中检测和定位热带念珠菌mRNA的表达。这项原位杂交研究揭示了首个直接证据，证明热带念珠菌分泌天冬氨酸蛋白酶2（CtSAP2）沉积在假菌丝尖端，并参与急性系统性念珠菌病。我们得出结论，原位RT-PCR可成功应用于FFPE组织，并将为研究念珠菌属中的基因表达提供新的视角。
