Biomark hd delta gene assay

Gene expression for selected targets identified from RNAseq analysis were examined in an independent set of microglial samples isolated from PND35 female littermates of animals used for sequencing (Supplemental Table S3). Gene expression analysis was performed using the Fluidigm Biomark HD Delta gene assay on a 48.48 Integrated Fluidic Circuit. Delta gene assay design was performed by Fluidigm and primer sequences are listed in Supplemental Table S3. 20ng of total RNA was used as input for cDNA synthesis (Fluidigm, 100–6472 B1) followed by pre-amplification (13ng per sample) (Fluidigm, 100–5875 C1) using a mix of all forward and reverse primers. Samples were then diluted five-fold and analyzed on a 48.48 IFC and Biomark HD machine (1.4ng/sample) in duplicate (Fluidigm, 100–9791 B1). Cycle threshold values were averaged across replicates and then normalized to hprt expression (no difference in mean Ct values for hprt between conditions, t= 1.27, p = 0.239) using the delta Ct method (Ct gene – Ct hprt). These values were then used for group comparisons with a two-tailed t-test without an assumption of equal variance. For display purposes, the delta delta Ct values were then calculated relative to the average Ct value of the PBS treated control for each gene, and relative expression levels were then calculated for each group by 2^-delta delta Ct (Figure S4D).