Quantitative analysis of mRNA expression of neuron-specific growth-associated genes in rat primary neurons by competitive RT-PCR.
The reverse transcriptase-polymerase chain reaction (RT-PCR) application is a sensitive method for detecting gene expression in tissues where the message level is a very small percentage of the total RNA and where only small amounts of sample are available such as in primary cultured hippocampal neurons. Based on a previously developed quantitative competitive RT-PCR strategy, mRNA expression and regulation of the neuron-specific growth-associated genes T alpha1 alpha-tubulin (T alpha1), microtubule-associated protein-2 (MAP-2) and growth-associated protein-43 (GAP-43), all of which have been proposed as putative markers of neurite growth during development and regeneration, were quantitated. This protocol, in combination with morphological evaluation of neurite outgrowth, may provide a useful tool for quantitation of neurite outgrowth during differentiation and regeneration in cultured neurons and may also be applied to detect the expression of other genes where the levels of message are low and in other tissues where small quantities of RNA are available.
Wang
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Department of Pediatrics, Kingston General Hospital, Room 6-301, Doran 3, Queen's University, Kingston, Ont. K7L 2V7, Canada.
Dow
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KE
D000818:Animals; D001667:Binding, Competitive; D002454:Cell Differentiation / Q000235:genetics; D019922:GAP-43 Protein / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D016015:Logistic Models; D008869:Microtubule-Associated Proteins / Q000235:genetics; D009416:Nerve Regeneration; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009474:Neurons / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D014158:Transcription, Genetic; D014404:Tubulin / Q000235:genetics
D019922:GAP-43 Protein; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D014404:Tubulin