Lightcycler 384 real time pcr system

Total RNA was extracted using a commercial kit (miRNeasy Mini Kit, Qiagen) according to the manufacturer's instruction, and the concentration was measured using a NanoDrop device. Total RNA extracts were diluted at 20ng/ml and reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and then diluted 1/2. Quantitative PCR reactions were performed using LightCycler DNA Green Master (Roche) on a LightCycler 384 Real-Time PCR System thermocycler (Roche). The amplification curves were analyzed by the LightCycler 480 SW 1.5 software. HPRT was amplified as qPCR normalization gene.

The total RNA of retinas or cells was isolated using the TRIzol reagent (Invitrogen). RNA from each sample was converted to cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). qRT-PCR was then performed using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme) in the Light Cycler^® 384 Real-Time PCR system (Roche Diagnostics). The RNA level of each gene was normalized to that of a β-actin gene in the same sample and relative RNA quantities were obtained using the comparative cycle threshold method (∆∆CT method). All used qRT-PCR primers are listed in Table S3.

RT-qPCR was performed on four RNA samples from each of the two groups to verify the accuracy of the RNA-seq data. Using the Prime Script™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China), total RNA was reverse transcribed into cDNA. Specific primers were devised with Primer 5.0 and were synthesized by Beijing Tsingke Biotech Co., Ltd., Beijing, China (Table 1). The qPCR was conducted by mixing 5 µL of 2× TB Green Premix Ex Taq II (Takara), 0.5 µL of each primer (forward and reverse), 200 ng of cDNA template, and RNase-free water up to 10 µL and run on a Light Cycler 384 real-time PCR system (Roche, Basel, Switzerland) with the following program: Stage 1: 95 °C for 30 s; Stage 2: 95 °C for 5 s and 60 °C for 30 s for 40 cycles; Stage 3: 95 °C for 5 s, 60 °C for 1 min and 95 °C for 5 s; and Stage 4: 50 °C for 30 s. The 2^−∆∆CT approach was utilized, and quantitative analysis was carried out. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for lncRNA, β-actin for mRNA and circRNA, and U6 for miRNA [20 ].