A sensitive quantification method for evaluating the level of hepatocyte growth factor and c-met/HGF receptor mRNAs in the nervous system using competitive RT-PCR.
Conventional RNA quantification methods such as Northern blots or RNase protection assays are often not sufficiently sensitive to measure mRNA levels in a small neuronal region. The reverse transcription-polymerase chain reaction (RT-PCR) is a sensitive alternative that can be used to determine the relative amount of mRNAs in tissues or cells. However, this method does not directly yield the absolute value of mRNA abundance because of the exponential nature of PCR. Using synthetic RNA competitors, we developed a competitive RT-PCR to evaluate the absolute amount of hepatocyte growth factor (HGF) and c-met/HGF receptor mRNAs in neural tissues. Here we describe the procedures we used to measure HGF and c-met mRNA levels in the punched ventral horn of the mouse spinal cord. This protocol provides a rapid, sensitive and accurate means of measuring mRNA levels and allows for comparison of the expression of related genes at one time and in a tiny piece of sample from a specific neuronal region.
Sun
W
W
Division of Biochemistry, Department of Oncology, Biomedical Research Center, B-7, Osaka University Medical School, Osaka, Japan.
Funakoshi
H
H
Matsumoto
K
K
Nakamura
T
T
D000818:Animals; D000870:Anterior Horn Cells / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009420:Nervous System / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013116:Spinal Cord / Q000378:metabolism
D012333:RNA, Messenger; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met