Quantitative RT-PCR Analysis of Murine Retina

Total RNA was isolated from murine retina and analysis was performed using an iQ5 Multicolor Real-time PCR I-cycler (Bio-Rad Laboratories, Hercules, CA) as described previously [15 (link)]. Reactions were performed with cDNA (6 ng/reaction, in triplicate) using 1X PCR buffer (Bioline), 3 mM MgCl₂, 10 nM fluorescein (USB Corporation), 0.1% Triton X-100, 0.03 U/μL Immolase DNA polymerase (Bioline), 800 μM dNTP mix (Bioline), 0.025X SYBR Green (Invitrogen), and 200 nM of forward and reverse primers in a total volume of 25 μL. Normalized gene expression was determined using the iQ5 optical system software (BioRad) using acidic ribosomal phosphoprotein P0 (ARBP) expression as a reference gene for each sample. The sequence for primers used in qRT-PCR is provided in Table 2.