Quantification of transcripts in testes preparation and its application to other tissue sections

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Testis was a suitable organ for quantifying the ISH signals because germ cells undergo synchronized development and show stage-specific gene expression. Previously, we used ribosomal RNA as the hybridizable RNA in paraffin sections and could easily analyze ISH signals expressed with digoxygenin-labeled probes quantitatively, through "posterization" of the images. In the present project, we applied this method to analyze the quantification of transcripts, PERF 15 mRNA. PERF 15 was a 15 kDa protein found in the perforatorium of the sperm head and identified as a.testis lipid binding protein. To determine an absolute quantity of transcripts in tissue sections, we further analyzed the signals by a Confocal Laser Scanning microscope with the use of a tyramide signal amplification system.The peak of PERF 15 mRNA expression was found in diplotene spermatocytes, and the amount of PERF 15 mRNA w as greatest in late pachytene and diplotene primary spermatocytes and early spermatids, followed by early pachytene primary spermatocytes, and then late spermatids. PERF 15 may be involved in the events leading to meiotic division, in which apoptosis is also involved.The present study could help our attempt to determine the concentration of mRNA in the tissue section. In this protocol, we made glass slides on which a tissue section and row of oligonucleotides spots were placed to determine the concentration of mRNA in a tissue section. These spots, 110 pm in diameter, consisted of 1 nl of sense and antisense mRNA sequences, from 0-1 × 10^<-6> M to 500 M, and we attempted to compare the signal intensity in the tissue section with the signal intensity in the spots containing known concentrations of the given mRNA.