M mlv reverse transcriptase

Total RNA was extracted from tumor tissues or cells using a simple total RNA kit (BioTeke, Beijing, China), and the RNA was then reverse transcribed into cDNA using the M-MLV Reverse Transcriptase (BioTeke, Beijing, China). Quantitative real-time PCR was performed using a SYBR-Green method (Takara, Beijing, China) in an Exicycler^TM 96 real time (RT)-PCR machine (Bioneer, Daejeon, Korea). GAPDH was used as an endogenous control for normalization. The primers used for this study included: SALL4, 5′-CCGCACTGAGATGGAAGGT-3′(forward), and 5′-GCTGGGCTGCTAACAAA-GG-3′(reverse); GAPDH, 5′-GAAGGTCGGAGTCAACG GAT-3′(forward), and 5′-CCTGGAAGATGGTGATGGGAT-3′ (reverse).

Total RNA was extracted from tissues and cells using the RNA pure Extraction Kit (BioTeke Corp., Beijing, China), according to the manufacturer's instructions. RNA sample concentration was measured by UV spectrophotometry and RNAs were reverse-transcribed using M-MLV Reverse Transcriptase (BioTeke Corp.). Primers were synthesized by Sangon Biotech Co., Ltd., (Shanghai, China) and the primer sequences were as follows: Notch1, forward 5′-TGGCTCCATCGTCTACCTG-3′ and reverse 5′-GGCTCCACCGTCTCACTCT-3′; Notch4, forward 5′-GGACTAGGAAATCCCGAACC-3′ and reverse 5′-AACCTCCCGAGCATCAGC-3′; Jagged1, forward 5′-GTGCCGCCATAGGTAGAGT-3′ and reverse 5′-CCAGCCAACCACAGAAAC-3′; Delta-like 1 (DLL1), forward 5′-GGGACGATGTTCGGATAA-3′ and reverse 5′-TCGGCACAGGTAGGAGTT-3′; Mastermind-like protein 1 (MAML1), forward 5′-AGCAACAGTTTCAGCGTCAT-3′ and reverse 5′-GCACAGCAGCAGAAGGTC-3′; p300, forward 5′-ATGATGCCTCGGATGACA-3′ and reverse 5′-GACACTGGTGCTTGACTGC-3′; and β-actin, forward 5′-GGAGATTACTGCCCTGGCTCCTAGC-3′ and reverse 5′-GGCCGGACTCATCGTACTCCTGCTT-3′. Amplification was carried out on an Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer Corp., Daejeon, Korea) and the amplification conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec. Results were normalized to β-actin and analyzed using the 2^−ΔΔCq formula (17 (link)).

The total RNA was extracted with a TRIpure kit (BioTeke, Beijing, China) and reversely transcribed into cDNA with M-MLV reverse transcriptase (BioTeke) in the presence of Oligo(dT) and random primers (Sangon, Shanghai, China).
After concentration measurement, the cDNA was used for real-time PCR with 2×Power Taq PCR Master Mix (BioTeke) and SYBR Green (Solarbio, Beijing, China) to detect the mRNA level of PPARγ, with β-actin as the internal control. The procedure was set as follow: 94°C for 5 min, 94°C for 10 sec, 60°C for 20 sec, 72°C for 30 sec, followed with 40 cycles of 72°C for 2 min 30 sec, 40°C for 1 min 30 sec, melting from 60°C to 94°C each 1°C for 1 sec, and finally incubated at 25°C for several minutes. The real-time PCR primers were purchased from Sangon, and the sequence information are shown in Table 1. The data was analyzed with 2^−ΔΔCt method.

Total RNAs were extracted from the retinal tissues using RNApure Total RNA Extraction kit (BioTeke, Beijing, China) and reverse-transcribed to cDNAs using M-MLV Reverse Transcriptase (BioTeke). The volume of obtained cDNAs was 20 µl. qPCR analysis was carried out following the reaction conditions on Real-Time PCR system (BIONEER, Daejeon, Korea): 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec. The primers used were as follows: NgR forward, 5′-GTCCCTTCCAGACCAATCAGC-3′ and reverse, 5′-GCCATTGCCTGGTGGAGTGT-3′; RhoA forward, 5′-TCGGAATGATGAGCACACAA-3′ and reverse, 5′-GCTTCACAAGATGAGGCAC-3′; Rock1 forward, 5′-GTGATGGCTATTATGGACG-3′ and reverse, 5′-AGGAAGGCACAAATGAGAT-3′; F-actin forward, 5′-GAAGAGAAAGCAGCAGTGTTA-3′ and reverse, 5′-GGAGCCAGAGGGTGGTTAT-3′; GAP-43 forward, 5′-AGGGAGATGGCTCTGCTAC-3′ and reverse, 5′-CACATCGGCTTGTTTAGGC-3′; GAPDH forward, 5′-CGGCAAGTTCAACGGCACAG-3′ and reverse, 5′-CGCCAGTAGACTCCACGACAT-3′. The primers were synthesized by Sangon Biotech. Gene expression was normalized to GAPDH and calculated using the 2^−ΔΔCq formula.