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Quantitative Fluorescent RT‐PCR Measurements of Postnatal Calcium Channel Gene Expression in Rat Hippocampal Subfields

ature function and activity of voltage-gated Ca channels is attained over the first several weeks of postnatal life. Previous work has described some aspects of these developmental changes. In hippocampal CA3 neurons, presynaptic Ca channels were predominantly N-type early in development and switched to mostly Q-type later in life. To learn more about the developmental regulation of Ca channel genes, we studied the postnatal expression of the α1 subunit, since this is the pore-containing portion of this heteromeric channel protein. We required a technique that afforded both regional localization and precise quantitation. While in situ hybridization allows regional localization of gene expression, it yields only semiquantitative results. We developed a novel quantitative fluorescent RT-PCR method that used a gene-specific deletion construct that coamplified with the cDNA for each channel gene. Thus, this gene construct could serve as an external standard and generate accurate copy numbers of the gene of interest. We compared the regional expression of the α1 subunit genes, rbA, rbB, rbC, and rbD, in three different hippocampal areas in the early postnatal period and in adulthood. Biweekly samples were taken in the first month of life since this is the period of most active growth and development of neurons and their connections. Wistar rats from the first day of life (P0) through adulthood (ages P7, 13, 16, 25, and 80) were anesthesized with halothane and decapitated. Transverse dorsal hippocampal slices were isolated and then sectioned with a McIlwain chopper at 400 μm. Punch biopsies were taken from the somatic layers of the CA1, CA3, and dentate gyrus (DG) subfields using sterile 200-μL filter pipette tips (tip diameter 1 mm). Samples were transferred directly to 1.5-mL centrifuge tubes containing 1 mL TRIzol reagent (Gibco BRL) and RNA was isolated using standard protocols. RNA samples were reverse-transcribed using MMLV-Reverse Transcriptase (GIBCO BRL), and published primer sequences for each of the four genes were used. Deletion products were constructed for the four gene fragments and served as the external standards for quantitation in the PCR reactions. Each deletion product had priming sites identical to those of the full-size PCR product, which ensured coamplification at the same efficiency and differed only in containing the deletion of known length.