Transferrin receptor tfrc

Retinas and inner ears were excised from mice by microdissection and lysed using a TissueLyser II (Qiagen, Denmark). Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and first-strand cDNA was produced using random decamer primers (Eurofins Genomics, Germany) and Superscript IV reverse transcriptase (Thermo Fisher, Waltham, MA). A reaction without reverse transcriptase served as a negative control to detect genomic contamination. qPCR was performed using TaqMan real-time PCR assays (Thermo Fisher) with the primers specific to NBCn1 mRNA transcribed from promoters P1 (MEAD-NBCn1), P2 (MERF-NBCn1), or both (total-NBCn1). The primer sequences are available in the Supplementary Material. Reference genes were ribosomal subunit S18 and transferrin receptor TFRC (Thermo Fisher). Reactions were performed using MX3000 P (Agilent, Santa Clara, CA) at 95 °C for 5 min, and then 50 cycles at 95 °C for 10 s, 56 °C for 20 s, and 72 °C for 30 s. The cycle threshold C_T was determined using the MxPro qPCR software that was supplied with the instrument. The expression of NBCs relative to a geometric mean from reference genes was calculated and changes in mRNA expression were determined. The calculated NBCn1 expression levels were then normalized to the mean value for total-NBCn1 transcripts in retinas from WT mice.