Quantifying Dixdc1 Transcripts in Mouse Retina

Fresh retinas were dissected from the eyes of anesthetized P1, P5, P10, and adult B6/J and A/J mice and placed into RNAlater (BioRad), taking care to avoid RNase contamination. Adult samples were comprised of both retinas from individual animals, while developmental samples were comprised of pooled retinas from mice in the same litter (∼3–4 mice). RNA was purified from each sample using an RNeasy Mini Kit (#74104; Qiagen), and single stranded cDNA was produced from these samples using an iScript cDNA synthesis kit with oligo (dT) and random primers (#1708890; BioRad). Sso Advanced Universal SYBR Green Supermix was used to determine the relative expression of full-length Dixdc1 transcripts (using primers directed to the common 3′UTR) to the amount of the truncated transcripts via qPCR. Adult and developmental samples were assessed separately. For each gene, every sample was run on a single plate in triplicate using the CFX96 qPCR thermal cycler (BioRad). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and β-2 microglobulin (β2m) were used as internal control genes for normalizing Dixdc1 expression to compare quantities across samples (as described in Keeley et al., 2012 (link)). Details for the qPCR primers are listed in Table 3.