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Semiquantification and localization of brain cytokine by in situ RT-PCR

原位 RT-PCR 半定量和脑细胞因子定位

基本信息

批准号：
07680835
负责人：
KAWAZOE Yoko
金额：
$ 1.34万
依托单位：
Tokyo Metropolitan Institute for Neuroscience
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680835/
关键词：
Experimental autoimmune in situ RT-PCR Cytokine encephalomyelitis

项目摘要

In order to know the interaction between astrocytes and microglia during the course of experimental autoimmune encephalomyelitis (EAE), we tried to localize the cytokine mRNA in the central nervous system (CNS) by in situ RT-PCR after amplification of cytokine mRNA on tissue. Frozen or paraffin-embedded sections were pretreated with proteinase K and mRNA on sections was reverse transcribed into cDNA with the SuperScript Preamplification System. cDNA was then amplified using a TGF-beta1-specific primer pair in a thermal cycler by either the direct or indirect method. In the direct method, cDNA was amplified in the presence of DIG-dUDP and integrated DIG was detected by immunostaining for DIG.In the indirect method, amplification was performed using unlabeled nucleotides followed by in situ hybridization with DIG-labeled TGF-beta1-specific probe.It was revealed that so-called " DNA repair artifact " in which DIG-dUDP was integrated into nicks of genomic DNA could not be eliminated in spite of several pretreatment procedures. As a result, all the nuclei on section were stained positively. On the other hand, such artifact was not observed in the indirect method. At the peak stage of EAE,many infiltrating inflammatory cells and microglia were stained positively. However, it was difficult to obtain stable staining partly because of poor fixation of amplified PCR products on tissue. In conclusion, optimal pretreatment procedures must be undertaken to obtain stable and specfic staining.

为了解实验性自身免疫性脑脊髓炎（EAE）发病过程中星形胶质细胞和小胶质细胞之间的相互作用，本研究采用原位RT-PCR技术，对EAE大鼠中枢神经系统（CNS）细胞因子mRNA进行了定位。冰冻切片或石蜡包埋切片经蛋白酶K预处理后，用SuperScript Preamplification System将mRNA反转录成cDNA。然后使用TGF-β 1特异性引物对在热循环仪中通过直接或间接方法扩增cDNA。在直接方法中，在DIG-dUDP存在下扩增cDNA，并通过DIG的免疫染色检测整合的DIG。在间接方法中，使用未标记的核苷酸进行扩增，然后用DIG标记的TGF-β 1特异性探针进行原位杂交。dUDP整合到基因组DNA的切口中，尽管经过几种预处理程序，也不能消除dUDP。结果，切片上所有细胞核均呈阳性染色。另一方面，在间接方法中未观察到这种伪影。在EAE高峰期，大量浸润的炎性细胞和小胶质细胞染色阳性。然而，很难获得稳定的染色，部分原因是扩增的PCR产物在组织上的固定不良。总之，必须采取最佳的预处理程序，以获得稳定和特异性染色。