Lunascrip rt supermix kit

Total lens RNA was isolated from four C57BL/6 mice and seven double KO mice by using the PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA, USA). The extracted RNA was eluted in RNase-free water and incubated with TURBO DNase (Ambion, Austin, TX, USA) for 30 minutes followed by precipitation with lithium chloride to remove genomic DNA contamination. Complementary DNA synthesis reactions were performed with total RNA (1 μg) by using the LunaScrip RT SuperMix Kit (New England Biolabs, Ipswich, MA, USA), according to the manufacturer's instruction. The expression of TMEM16A, TMEM16B, and β-actin was determined using PCR. The cDNA samples were amplified using the GoTaqColorless PCR Master Mix Kit (Promega, Madison, WI, USA) on the Applied Biosystems 2720 Thermal Cycler (Forster City, CA, USA) with the strategy previously described by Kamikawa et al.9 (link) Briefly, cDNA (20 ng) and gene-specific primers pairs (Table 2) were added to a GoTaqColorless PCR Master Mix Kit and subjected to PCR amplification (1 cycle at 95°C for 2 minutes and 40 cycles at 95°C for 40 seconds, 55°C for 45 seconds, and 73°C 1 minute), followed by a dissociation stage (1 cycle at 73°C for 5 minutes). All PCR reactions were run in triplicates.