Neuromodulatory co-expression in cardiac vagal motor neurons of the Dorsal Motor Nucleus of the Vagus [PCR]

In this study we performed high throughput microfluidic qPCR using BioMark on RNA extracted from single neurons microdissected from DMV and NA tissue of one male Sprague Dawley rat. High throughput microfluidic qPCR using BioMark was performed on RNA extracted from 81 single neurons microdissected from DMV and NA tissue of one male Sprague Dawley rat. Two 96 x 96 BioMark Arrays were used to assay expression of 173 genes. A serial dilution gene set was included in each chip to confirm reproducibility. Technical replicates were run on each chip to evaluate technical variability. Data Normalization: Initial quality control included melt-curve analysis and removal of samples and genes with >30% failed reactions. Genes with >75% working reactions were used to generate a median expression level across all samples. Raw Ct values were normalized against this median expression level to obtain DCt values. DCt values were generated for each gene according to the following equation: DCt_gene = Ct_gene - (median sample Ct). The median across all samples within a gene was then used to calculate -DDCt from DCt values by use of the following equation: -DDCt_gene = -(DCt_gene - DCt_median_across_samples).