Sybr premix kit

Total RNA or miRNA was isolated and extracted by TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) or miRcute Extraction and Separation of miRNAs kit (Tiangen Biotech Co., Ltd., Beijing, China), and then reversely transcribed into cDNA using PrimeScript™ II 1st strand cDNA synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China). SYBR Premix kit or SYBR PrimeScript miRNA RT-PCR kit (both from Takara Biotechnology Co., Ltd.) was used for qRT-PCR. The thermocycling conditions were one cycle of initial denaturation at 95 °C for 3 min, 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Glyceraldehyde-3-phosphate or dehydrogenase (GAPDH) and U6 small nuclear RNA (U6) were used for normalization. The relative expression levels of miRNA and mRNA between the experimental group and the control group were calculated using 2-ΔΔCq method. The experiments were repeated at least 3 times. The primers were as follows: miR-122-5p forward, 5′-TATTCGCACTGGATACGACACAAAC-3′ and reverse, 5′-GCCCGTGGAGTGTGACAATGGT-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; CCNG1 forward, 5′-GTTACCGCTGAGGAGCTGCAGTC-3′ and reverse, 5′-GCAGCCATCCTGGATGGATTCAG-3′; GAPDH forward, 5′-GGTGAAGGTCGGAGTCAACG-3′ and reverse, 5′-CAAAGTTGTCATGGATGHACC -3′.

Total RNA from RF/6A or the posterior part of the eyeball (the retina, RPE and choroid) at 24 h after laser treatment was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) and then was reverse transcribed with PrimeScript RT Master mix (Takara, Otsu, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using an Eppendorf Mastercycler ep realplex machine (Eppendorf, Germany) and a SYBR Premix Kit (Takara), according to the manufacturers’ instructions. The sequences of the specific primers were presented in Table 1. Relative mRNA expression levels were calculated by the 2^-△△Ct method. GAPDH was used as a reference gene.

MiRNAs expression levels were assayed by real-time PCR using a SYBR premix kit (TaKaRa) according to the manufacturer’s protocol. Total RNAs extracted from lVt (GV)- and MI-stage were reverse transcribed using M-MLV reverse transcriptase (Promega) and a stem-looped antisense primer. The resultant cDNA was submitted to the amplification of mature miRNAs using a miRNA specific primer and a universal primer. U6 snRNA gene was employed as an endogenous control. The primers for miRNAs and U6 snRNA are listed in Additional file 4: Table S3). Quantitative real-time PCR was conducted in 25 μl reaction volumes containing 300 nM of each primer and cDNA derived from 0.1 μg of total RNA. Cycling parameters were 95°C for 3 min, and followed by 35 cycles of 95°C for 35 s and 60°C for 30 s. All reactions were run in triplicate. The expression of miRNAs was normalized against U6 snRNA levels. The data are presented as means ± SE. Statistically significant differences were examined by paired t-test. A value of P < 0.05 was considered to be statistically significant.

The process of RT-qPCR was reported in our previous study [51 (link)]. Briefly, total RNA was isolated from PLCs using an RNA extraction reagent kit (TaKaRa Bio, Inc., Dalian, China). cDNA was synthesized using a reverse transcription reagent kit (TaKaRa Bio, Inc., Dalian, China). RT-qPCR was performed with a Roche Light Cycler 480 II PCR instrument using a SYBR premix kit (TaKaRa Bio, Inc., Dalian, China) according to the manufacturer’s instructions. The sequences of the specific primers are listed in Table 2. Each gene was repeated three times as technical replicates. The 2^−ΔΔCt method was used to perform the gene mRNA quantifications, and β-actin was used as an internal control gene to normalize the amount of transcripts in each sample to correct for differences in the cDNAs used.