1996 Fiscal Year Final Research Report Summary
Semiquantification and localization of brain cytokine by in situ RT-PCR
| Project/Area Number |
07680835
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Nerve anatomy/Neuropathology
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| Research Institution | Tokyo Metropolitan Institute for Neuroscience |
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Principal Investigator |
KAWAZOE Yoko Tokyo Metropolitan Institute for Neuroscience, 神経病理研究部門, 主任研究員 (60281705)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUMOTO Yoh Tokyo Metropolitan Institute for Neuroscience, 神経病理研究部門, 副参事研究員 (90173921)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Experimental autoimmune / in situ RT-PCR / Cytokine / encephalomyelitis |
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| Research Abstract |
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.
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