Quantification of neurotrophin-3 mRNA in the rat hippocampal subregions using the RT-PCR-based coamplification method.
Quantitative reverse transcription-polymerase chain reaction (RT-PCR) is a suitable method for determining the expression levels of rare mRNAs in small amounts of tissue. To compare the mRNA expression levels across specific brain regions, we adopted an RT-PCR method in which a target gene was coamplified with an endogenous internal standard gene in single reaction tubes. Use of the endogenous internal standard can control fluctuations in target quantification resulting from various factors, including tube-to-tube variation in amplification efficiency and variation in mRNA content among the total RNAs prepared from different tissues. In this study, we quantitatively determined the mRNA expression levels for NT-3, a member of the neurotrophin family of growth factors, in the hippocampal subregions: the entorhinal cortex, dentate gyrus and CA1. NT-3 gene was simultaneously coamplified with an endogenous internal standard gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT), in the same reaction tube. Using this RT-PCR coamplification method, we detected a regional difference in the NT-3 mRNA expression levels across the hippocampal subregions. Our method can serve as a useful quantification method to investigate molecular signaling cascades in a specific cortical region.
Tokuyama
W
W
Department of Physiology, University of Tokyo School of Medicine, Hongo 7-3-1, Bunkyo-ku, Tokyo, Japan. tokuyama@m.u-tokyo.ac.jp
Hashimoto
T
T
Li
Y X
YX
Okuno
H
H
Miyashita
Y
Y
D000818:Animals; D015152:Blotting, Northern; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D006624:Hippocampus / Q000737:chemistry* / Q000201:enzymology; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D020933:Neurotrophin 3 / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
D017931:DNA Primers; D018076:DNA, Complementary; D020933:Neurotrophin 3; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase