Primescript rt master mix kit

Total mRNA was extracted using TRIzol reagent (CWBIO, CW0580S, Beijing, China) and RNA concentrations were determined using an ultraviolet spectrophotometer. cDNA was performed using a Prime Script™ RT Master Mix Kit (TransGen, AE301-02, Beijing, China) according to the manufacturer’s instructions. qPCR was performed using an Ultra SYBR Mixture Kit (CWBIO, CW0957C, Beijing, China) and a Bio-Rad CFX Maestro system. The reaction parameters were as follows: 95 °C for 15 min, 45 cycles of amplification with three steps: denaturation at 95 °C for 10 s, annealing at 55 °C for 20 s and extension at 72 °C for 30 s.
The primers used are listed below:
GAPDH forward 5′ AACGGATTTGGTCGTATTGG and
GAPDH reverse 5′ GGCTGCTGTCACCCATGAA
SPHK1 forward 5′ AACGGATTTGGTCGTATTGG and
SPHK1 reverse 5′ TCACTCTCTAGGTCCACATCAG
PBX1 forward 5′ CAGTGAGGAAGCCAAAGAGG and
PBX1 reverse 5′ CAGCTGTTTTGGCAGCATAA
S1PR1 forward 5′ GCCTACACAGCTAACCTGCTCTTG and
S1PR1 reverse 5′ TGGCGATGGCGAGGAGACTG
S1PR2 forward 5′ CCACCACCTCCTGCCACTCC and
S1PR2 reverse 5′ CACCGTGTTGCCCTCCAGAAAC
S1PR3 forward 5′ GATCCTCTACGCACGCATCTACTTC and
S1PR3 reverse 5′ ACACGCTCACCACAATCACCAC
S1PR4 forward 5′ GAAGCCGTAGACGCGGCTGG and
S1PR4 reverse 5′ GAAGCCGTAGACGCGGCTGG
S1PR5 forward 5′ GTGAGGTGGGAGCCATAGAA and
S1PR5 reverse 5′ TTGGCTGAGTCTCCCAGAGT

Total RNA from cells was extracted using RNAiso Plus reagent (TAKARA, China), according to the manufacturer's instructions. Total RNA was transcribed into cDNA using a PrimeScript RT Master Mix kit (TransGen Biotech, China). The real-time quantitative PCR reaction was performed using the TransStart Tip green qPCR SuperMix kit (TransGen Biotech, China) in an ABI 7500 Real Time PCR System (Applied Biosystems, USA). Primer sequences were detailed in Table S1. The β-actin or GAPDH gene was amplified separately as an internal control to normalise for specific gene expression in the samples. Fold change was calculated using the 2^-DDCt method.

Total RNA was extracted from cells using RNAiso Plus reagents (TAKARA, China), according to the manufacturer's instruction. Total RNA was transcribed into cDNA using a PrimeScript RT Master Mix kit (TransGen Biotech, China). The real-time quantitative PCR reaction was performed using a TransStart Tip green qPCR SuperMix kit (TransGen Biotech, China) in an ABI 7500 Real Time PCR system (Applied Biosystems, USA). Primer sequences were detailed in Table s1 and s2. The β-actin gene was amplified separately as an internal control to normalize for specific gene expression in the samples. The fold change was calculated using the 2^-∆∆Ct method.