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

睾丸是一个合适的器官定量ISH信号，因为生殖细胞进行同步发展，并显示阶段特异性基因表达。以前，我们使用核糖体RNA作为石蜡切片中的可杂交RNA，并且可以通过图像的“后处理”来容易地分析用地高辛标记的探针定量表达的ISH信号。在本项目中，我们应用这种方法来分析转录本，PERF 15 mRNA的定量。PERF 15是在精子头部的穿孔管中发现的15 kDa蛋白，并被鉴定为睾丸脂质结合蛋白。为了确定组织切片中转录物的绝对量，我们进一步通过使用酪胺信号放大系统的共聚焦激光扫描显微镜分析信号，发现PERF 15 mRNA在双线期精母细胞中表达最高，并且PERF 15 mRNA的量在晚粗线期和双线期初级精母细胞以及早期精子细胞中最高，其次是早粗线期初级精母细胞，然后是晚期精子细胞PERF 15可能参与了减数分裂的过程，同时也参与了细胞凋亡的过程，本研究有助于我们确定组织切片中mRNA的浓度。在该方案中，我们制作了载玻片，在载玻片上放置组织切片和一排寡核苷酸点，以测定组织切片中mRNA的浓度。这些斑点直径为110 μ m，由1 nl正义和反义mRNA序列组成，从0-1 × 10^<-6>M到500 M，我们试图将组织切片中的信号强度与含有已知浓度给定mRNA的斑点中的信号强度进行比较。

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.