Takara reverse transcriptase

Total RNA was extracted using the Trizol reagent, according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). 2 ug of total RNA were reverse transcripted using TaKaRa reverse transcriptase (TaKaRa Biotechnology, DaLian, China). A volume of 2.0 ul of each diluted cDNA (1∶20) was subjected to Real-time quantitative PCR in a final volume of 20 ul containing 100 nm of each specific primer and 1×SYBR Green Mix (TaKaRa). The sequences of KLF4 and β-actin primers were as follows: KLF4 gene, F: 5′-aagagttcccatctcaaggcaca-3′, R: 5′-gggcgaatttccatccacag-3′ and β-actin gene, F: 5′-ctaagtcatagtccgcctagaagca-3′, R: 5′tggcacccagcacaatgaa-3′. The amplification was carried out as follows: initial emzyme activation at 95°C for 30 s, then 40 cycles of 95°C for 5 s, 60°C for 30 s, and then a dissociation stage using an iQ5 multicolor real-time PCR Detection System (Bio-RAD, Hercules, CA). The cycle threshold (CT) value was determined as the point at which the fluorescence exceeded a threshold value preset by the instrument’s software. Relative expression of KLF4 in each experiment set (fold-change to control) was calculated according to comparative Ct method using the formula: RQ = 2^−ΔΔCt.

Total RNA was respectively extracted from the retinas in three subgroups (n = 6 per subgroup, every two eyeball specimens in each group were pooled) using trizol reagent (Beyotime, Biotechnology, China) according to the manufacturer’s protocol. This was followed by reverse-transcription of 1 μg of total RNA to cDNA using Takara reverse transcriptase and the random primer provided in the kit (Takara, Japan). QPCR was performed on a Roche LightCycler instrument with Takara SYBR green II kit using resultant cDNA as template. The primer sequences were listed in Table 1. The protocol for the qPCR was an initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 59°C for 60 s, and extension at 97°C for 1 s. At the end of the amplification, calculating the relative expression of target genes in each group with the 2^–△△ CT method (Ding et al., 2020 (link); Wu et al., 2020 (link)).