Non-cloning Synthesis of cRNA Probe and Its Application to In Situ Hybridization Analysis of Formalin-fixed Paraffin Embedded Tissue

Abstract

Intestinal type alkaline phosphatase (IALP) mRNA-specific cRNA probe was synthesized by non-cloning method for in situ hybridization (ISH) analysis. Total RNA was extracted from small intestine of male F344 rat. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for IALP gene. T7 promoter adapter was directly added to IALP cDNA-specific PCR product by ligation reaction. IALP cDNA accompanied by T7 promoter adapter was then amplified by PCR using IALP gene-specific primer in combination with T7 promoter adapter primer sequence and used as a template of cRNA probe. Digoxigenin-labeled cRNA was synthesized by in vitro transcription method using RNA polimerase. ISH was performed using formalin-fixed, paraffin embedded small intestine tissue. As a result, the cytoplasm of mucosal epithelial cells was successfully stained with this cRNA probe. The present procedure is quite simple and a rapid way to prepare the cRNA probe and would be very useful for ISH analysis of formalin-fixed paraffin embedded tissue sample.