Abstract
Fluorescence-based PCR techniques are becoming an increasingly popular method for measuring low-abundance alternatively spliced mRNA transcripts. The dynamic range of real-time RT-PCR affords high sensitivity for the measurement of gene expression, but this mandates the need for strict controls to ensure assay validity. Primer design, reverse transcription, and cycling conditions need to be optimized to ensure an accurate and reproducible assay. Here, we describe a procedure for creating a cost effective and reliable method for the absolute quantification of several exon-skipping variants of human excitatory amino acid transporter-2 (EAAT2). We show that the cycling conditions can be adjusted to increase the specificity of primers that span exon–exon junctions, and that differences in the reverse transcription reaction can be minimized. Standard curves are stable and produce accurate absolute copy number data. We report that exon-skipping transcripts, EAAT2Δ7 and EAAT2Δ9, account for 5.8% of EAAT2 mRNA in autopsy human neocortex.
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