Rotor gene 3000 real time pcr instrument

Total RNA was extracted from specific tissues and cells using TRIZOL reagent (Thermo Fisher Scientific). RNA was reverse transcribed into cDNA on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) using a RevertAidTM H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed on a Rotor-Gene 3000 Real-time PCR instrument (Corbett Research, Sydney, Australia). The PCR program of miRNA-210 was 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The PCR program of EGR3 was 95 °C for 10 min, 35 cycles at 94 °C for 30 s, 53 °C for 45 s and 72 °C for 45 s. U6 was used as an internal reference for miRNA-210, and GAPDH was used as an internal reference for EGR3. The primers sequences were shown as follows: miRNA-210-F: 5′-GTGCAGGGTCCGAGGT-3′, miRNA-210-R: 5′-TATCTGTGCGTGTGACAGCGGCT-3′; U6-F: 5′-CTCGCTTCGGCAGCAC-3′, U6-R: 5′-AACGCTTCACGAATTTGCG-3′; EGR3-F: 5′-TACAATCAGATGGCTACAGAGAAT-3′, EGR3-R: 5′-TTCCCAAGTAGGTCACGGTC-3′; GAPDH-F: 5′-TCGGAGTCAACGGATTTGGTC-3′, GAPDH-R: 5′-GCCATGGGTGGAATCATATTGG-3′. Data was calculated in accordance with the 2^-∆∆Ct method.

The total RNA was extracted with TRIzol according to the manufacturer’s instructions. Following quantification by UV spectrophotometry, 1 μg of the total RNA was used for reverse transcription reaction synthesis of cDNA with a reverse transcription kit, according to the manufacturer’s instructions. PCR was used to amplify the cDNA. The corresponding primer sequences were as follows: forward, 5′-CCACCCATGGCAAATTCCATG-3′ and reverse, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′ for reference GAPDH; and forward, 5′-TTGAAGACCATAACCCACCA-3′ and reverse, 5′-CACATAGCGCCTCTGACTG-3′ for PTEN.
Quantitative PCR was performed using a Rotor-Gene 3000 Real-Time PCR instrument (Corbett Research, Australia). PTEN and β-actin mRNA were amplified by SYBR-Green real-time PCR using the One Step PrimeScript RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China). GAPDH mRNA was used as the internal control. The reactions used the following cycling conditions: 94°C initial denaturation for 3 min, 94°C denaturation for 30 sec, 60°C annealing for 30 sec and 72°C extension for 30 sec for a total of 35 cycles, and a final extension at 72°C for 7 min. Relative PTEN mRNA expression levels normalized to those of β-actin mRNA were calculated using the equation: 2^−ΔΔCt, where ΔΔCt(relative quantification) = (CT_PTEN - CT_β-actin)_{CHB patient} - (CT_PTEN - CT_β-actin)_{Normal control}

Total RNA was extracted from the frozen retina tissue by using Trizol reagent according to the manufacturer's instructions. cDNA synthesis was conducted according to the RNA PCR kit protocol (Takara, Dalian, China). ß-actin was used as a normalizing control. The primer sequences were 15-LOX-1: 5′-TTG GTT CTA CTG GGT TCC TAA TG-3′ (forward) and 5′-GGA GCC AAA CGA CAT TTA TCT G-3′ (reverse); PPAR-γ: 5′-ATG GAG CCT AAG TTT GAG TTT G-3′ (forward) and 5′-CAG CAG GTT GTC TTG GAT GTC-3′ (reverse); VEGFR-2: 5′-TCG AGC CCT CAT GTC TGA AC-3′ (forward) and 5′-TGA TGC TGT CCA AGC GTC TT-3′ (reverse); ß-actin: 5′-CTG AGA GGG AAA TCG TGC GT-3′ (forward) and 5′-CCA CAG GAT TCC ATA CCC AAG A (reverse). The PCR reaction was performed in a volume of 25 µl using SYBR green mix (Toyobo, Shanghai, China) on the Rotor-Gene 3000 Real-time PCR instrument (Corbett Research, Sydney, Australia). The thermal cycling program consisted of 1 minute at 95°C, 40 cycles of 15 seconds at 95°C, 15 seconds at 58°C, 45 seconds at 72°C. Relative quantification of the gene expression was performed using the 2^(−ΔΔCT) method [34] (link). All experiments were carried out three times.