Total RNA was purified from mouse retinas using Sepasol RNA I Super G (Nacalai Tesque, Japan), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Japan). A quantitative polymerase chain reaction (qPCR) was performed by the SYBR Green-based method with the Roche Light Cycler 96 (Roche Diagnostics, Japan). Gapdh was used as an internal control, and primer sequences were as previously reported (Kuribayashi et al., 2018 (link)). To examine the knockdown efficiency of shRNA-expressing plasmids, a total of 100 μg of plasmids containing 80 μg of pU6-shNmnat2 or pU6-Scramble and 20 μg of enhanced green fluorescent protein (EGFP)–expressing plasmids (pCAG-EGFP) were electroporated into NIH3T3 cells with electroporator CUY-21 (Nepa Gene, Japan). After 1–2 d, NIH3T3 cells were treated with trypsin and filtered, and EGFP-positive cells were collected by a cell sorter, FACS Aria SORP (BD Biosciences, USA). Total RNA was purified from the sorted cells, and cDNA was synthesized. Knockdown efficiency of shNmnat2 was examined by qPCR using primer for Nmnat2. Effects of shNmnat2 for the expression of Nmnat1 and Nmnat3 were also examined by RT-qPCR using primers for Nmant1 or Nmnat3.