A modified in situ RT-PCR method for localizing fungal-specific gene expression in Candida-infected mice renal cells.
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.
Yong
Voon Chen
VC
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra Malaysia, Serdang, Selangor, Malaysia.
Ong
Khang Wei
KW
Sidik
Shiran Mohd
SM
Rosli
Rozita
R
Chong
Pei Pei
PP
D000818:Animals; D057055:Aspartic Acid Proteases / Q000235:genetics; D041022:Candida tropicalis / Q000235:genetics* / Q000472:pathogenicity; D002177:Candidiasis / Q000382:microbiology*; D005260:Female; D015870:Gene Expression; D005800:Genes, Fungal; D017403:In Situ Hybridization / Q000379:methods*; D007668:Kidney / Q000382:microbiology; D007674:Kidney Diseases / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010336:Pathology / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D057055:Aspartic Acid Proteases