Multiplex semi-quantitative reverse transcriptase–polymerase chain reaction of low abundance neuronal mRNAs

• Published in: Brain research. Brain research protocols 1999-12, Vol.4 (3), p.395-406
• Main Authors: Pernas-Alonso, Roberto, Morelli, Francesco, di Porzio, Umberto, Perrone-Capano, Carla
• Format: Article
• Language: English
• Subjects: Animals; Brain primary culture; Carrier Proteins - genetics; Cells, Cultured; DNA Primers; Dopamine - genetics; Dopamine Plasma Membrane Transport Proteins; Dopaminergic neuron; Female; Gene Expression; HPRT; Hypoxanthine Phosphoribosyltransferase - genetics; Male; Membrane Glycoproteins - genetics; Membrane Transport Proteins; Mice; Mice, Inbred Strains; Mice, Neurologic Mutants; Motoneuron; Motor Neurons - chemistry; Motor Neurons - cytology; Motor Neurons - enzymology; Nerve Tissue Proteins; Neural gene expression; Neurofilament Proteins - genetics; Neurological mutant; Neuropeptides; Pregnancy; Rats; Rats, Sprague-Dawley; Reference Standards; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - standards; RNA, Messenger - analysis; Tyrosine 3-Monooxygenase - genetics; Vesicular Biogenic Amine Transport Proteins
• ISSN: 1385-299X
• DOI: 10.1016/S1385-299X(99)00045-8

The sequential use of reverse transcriptase and the polymerase chain reaction (RT-PCR) has provided molecular biology research with an exquisitely sensitive and fast technique for studying gene expression. This method is particularly useful to study transcripts in the nervous system, which are on average present at low levels and the amount of tissue or cells to be analyzed is often limited. Here, we describe a RT-PCR assay which allows the simultaneous detection and semi-quantitation of several transcripts (multiplex). Multiple PCR primer pairs are used to detect different target transcripts in a single reaction, together with a pair of primers able to amplify the hypoxantine-phosphoribosyl-transferase (HPRT), a gene constitutively expressed at low levels throughout the nervous system. HPRT levels remain constant also during neurogenesis and it is thus apt to be used in developmental neurobiology. This internal standard is the mRNA of reference to evaluate sample variation in RT and PCR reactions and to monitor the degradation and recovery of RNAs. Normalization with respect to HPRT cDNA allows to estimate the relative abundance of each target mRNA. Theme: Cellular and molecular biology Topic: Gene structure and function: general