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

睾丸制备中转录物的量化及其在其他组织切片中的应用

• 批准号: 18591801
• 负责人: SASAKI Junzo
• 金额: $ 2.51万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591801/
• 关键词: Anatomy; Cell and tissue; Testis; In situ hybridization; Gene; mRNA; PERF 15; Quantification of signals; PERF15; 核酸; 定量化

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.

睾丸是一个合适的量化ISH信号的器官，因为生殖细胞经历了同步发育并表现出阶段特异性的基因表达。以前，我们使用核糖体RNA作为石蜡切片中的杂交RNA，通过图像的“后处理”，可以很容易地定量分析地高辛标记的探针表达的ISH信号。在本项目中，我们应用这种方法来分析转录产物PERF 15mRNA的定量。PERF-15是在精子头部穿孔器中发现的一种15 kDa的蛋白质，被鉴定为睾丸脂结合蛋白。为了确定组织切片中转录本的绝对数量，我们利用酪酰胺信号放大系统对信号进行了进一步的分析，发现PERF 15mRNA在双线期精母细胞中的表达达到峰值，在粗线期晚期、双线期初级精母细胞和早期精子细胞中PERF 15mRNA的表达最高，其次是粗线期初级精母细胞，然后是晚期精子细胞。PERF 15可能参与了导致减数分裂的事件，而细胞凋亡也参与了减数分裂。本研究可能有助于我们确定组织切片中的mRNA浓度。在该方法中，我们制作了玻片，在玻片上放置组织切片和一排寡核苷酸斑点，以确定组织切片中的mRNA浓度。这些斑点直径110 PM，由1nL的正义和反义mRNA序列组成，范围从0-1×10~(-6)M到500M，我们试图将组织切片中的信号强度与含有已知浓度的给定mRNA的斑点中的信号强度进行比较。

项目成果

6-hydorxydopamineによるPC12細胞の細胞死とα-リポ酸(ALIPURE)の抑制機構

6-羟基多巴胺引起PC12细胞死亡及α-硫辛酸(ALIPURE)的抑制机制

• 发表时间: 2006
• 期刊: 医学と薬学 55.6
• 作者: Takashi;Kogami;Yukari;Mild;Teruo;Yamada;Teruo;Umegaki;Makoto;Nishimura;Takashi;Amo;Jun;Kosaka;Junzo;Sasaki;藤田洋史
• 通讯作者: 藤田洋史

Regulation of of 5-aminolevulinic acid-mediated protoporphyrin IX-accumulation and its photodynamic action in U937 cells.

5-氨基乙酰丙酸介导的原卟啉 IX 积累的调节及其在 U937 细胞中的光动力作用。

• 发表时间: 2007
• 作者: Hirofumi Fujita;et. al.
• 通讯作者: et. al.

Flow cytometric analysis of Ca^<2+>-induced membrane permeability transition of isolated rat liver mitochondria.

Ca ^ 2 诱导的离体大鼠肝线粒体膜通透性转变的流式细胞术分析。

• 发表时间: 2008
• 期刊: Journal of Clinical Biochemistry and Nutrition 42
• 作者: Teruo Umegaki;et. al.
• 通讯作者: et. al.

4-Hydoroxy-3,5,3',4'-tetrachlorobiphenyl induced membrane permeability transition in isolated rat liver mitochondria

4-羟基-3,5,3,4-四氯联苯诱导离体大鼠肝线粒体膜通透性转变

• 发表时间: 2006
• 期刊: J. Clin. Biochem. Nutr. 38
• 作者: Takashi;Kogami;Yukari;Mild;Teruo;Yamada;Teruo;Umegaki;Makoto;Nishimura;Takashi;Amo;Jun;Kosaka;Junzo;Sasaki;藤田洋史;Hirofumi Fujita
• 通讯作者: Hirofumi Fujita

Alpha-Lipoic acid suppresses 6-hydroxydopamine-induced ROS generation and apoptosis through the stimulation of glutathione synthesis but not by the expression of heme oxygenase-1.

α-硫辛酸通过刺激谷胱甘肽合成而非血红素加氧酶-1 的表达来抑制 6-羟基多巴胺诱导的 ROS 生成和细胞凋亡。

• 发表时间: 2008
• 期刊: Brain Res. 1206
• 作者: Fujita;H.;Shiosaka;M.;Ogino;T.;Okimura;Y.;Utsumi T.;Sato;E.;Akagi;R.;Inoue;M.;Utsumi;K.;Sasaki;J.
• 通讯作者: J.