Method for quantitation of low-abundance nerve growth factor mRNA expression in human nervous tissue using competitive reverse transcription polymerase chain reaction.
Nerve growth factor (NGF) is a polypeptide hormone important for the development, function, and survival of neurons. NGF is an important target for investigation because of its potential therapeutic importance in neurological diseases and injury, yet its low level of expression in nervous tissue makes it difficult to measure. We report here the development of a quantitative assay for human NGF mRNA, using competitive reverse transcription polymerase chain reaction (cRT-PCR), with mouse submandibular gland mRNA as an internal standard. Exhibiting high homology to the human transcript, mouse NGF mRNA is co-amplified with the human NGF transcript through both the reverse transcription and PCR steps using identical primers and conditions. Products are distinguished by a convenient unique restriction site in the mouse transcript. Phosphorimage detection and quantitation of radiolabeled product provides femtogram sensitivity. Generation of a standard curve from a dilution series of mouse mRNA allows the amount of co-amplified human NGF mRNA transcripts to be determined. We demonstrate the amplification and quantitation of human NGF mRNA from 175-350 nanograms of poly(A)+mRNA from human frontal and parietal cortex. This technique is ideal for rapid detection and quantitation of low-abundance NGF mRNA from limited amounts of tissue.
St Amand
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Department of Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
Pottage
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Henry
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Fahnestock
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M
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015252:Deoxyribonucleases, Type II Site-Specific; D005625:Frontal Lobe / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000235:genetics*; D010296:Parietal Lobe / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D013363:Submandibular Gland
D009414:Nerve Growth Factors; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D012194:RNA-Directed DNA Polymerase; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific