Stepone plus real time quantitative pcr system

Total hepatic RNA was extracted by a commercial RNA extraction kit (RNAiso Plus, Code No. 9108) provided by Takara Biomedical Technology (Beijing, China) Co., Ltd., and then reverse-transcribed into cDNA using a commercial cDNA kit with gDNA Eraser [Code No. RR047A] (Takara, Beijing, China). qPCR was completed in StepOne Plus Real-Time quantitative PCR System (Applied Biosystems, Foster City, CA, USA) with SYBE Green Ex Taq™ II [Tli RNaseH Plus, Code No. RR820A] (Takara, Beijing, China). The PCR conditions were as following: initial activation 95 °C for 30s, denaturation 95 °C for 5s, annealing 55 °C for 30 s, extension 72 °C for 30 s, 40 cycles. The mRNA expressions level was normalized to β-Actin gene. In this study, the 2^−ΔΔCT method was used to analyze the relative expression levels of related genes. The qPCR primers used in this study were purchased from Shanghai Sangon Biotech. Co., Ltd. (Shanghai, China) (Table 1).Table 1

Primer sequence for quantitative real-time PCR.

Table 1

Gene	Forward primer (5′-3′)	Reverse primer (5′-3′)
LDLr	TGGCTATGAGTGCCTATGTCC	GGTGAAGAGCAGAAACCCTATG
BSEP	CGTGCTTGTGGAAGAAGTTG	GGGAGTAGATGGGTGTGACTG
HMGCR	AGTGGTGCGTCTTCCTCG	CGAATCTGCTGGTGCTAT
CD36	GACAATCAAAAGGGAAGTTG	CCTCTCTGTTTAACCTTGAT
SREBP-1c	GCTGTTGGCATCCTGCTATC	TAGCTGGAAGTGACGGTGGT
CYP7A1	CACCATTCCTGCAACCTTTT	GTACCGGCAGGTCATTCAGT
β-Actin	ACGTCGACATCCGCAAAGACCTC	TGATCTCCTTCTGCATCCGGTCA

Total RNA was isolated using TRIzol reagent (Takara, Dalian, China). After synthesis from the RNA using HiScript® II Q Select RT SuperMix (Vazyme, Nanjing, China), cDNA was subjected to qRT-PCR amplification using a StepOne Plus Real-Time Quantitative PCR System (Applied Biosystems, CA, USA). The primers were synthesized by Bioengineering (Shanghai, China). Primer information is shown in Table 1. The relative level of each transcript was normalized to that of GAPDH and analysed according to the 2^−ΔΔCt method [19 (link)].Table 1

Real-time PCR primer sequences

Genes	Primer sequences (5′→3′)
ACAT2	F: TAATGATGGTGCTGCTGCTGTGG R: GCTTGCTTTATTGCCGGGATTGG
HMGCS1	F: AAGCACAGCCACCGAGCATATTC R: ACCATCCCACCCCACACTGAAG
HMGCR	F: TGTGATTGGAGTTGGCACCATGTC R: ACACGCAAGCTGGGAAGAAAGTC
MVK	F: GTTGTCTCAAGTCCTGCTGGTGTC R: AGGCTCACTTTCCCACTGTTGTG
PMVK	F: GGTGGATGATGCTGAGTCAGAGTG R: GTGCTGCTCATCTCCGTGGTTC
MVD	F: GCCACCTGCTTGGACACCTTC R: GGCGAAGATCACGGCGTTGG
FDFT1	F: GCGTCCACCCTCCTCACTCC R: CCCACACAGCCAGAGCCAAAG
SQLE	F: TGTGGACCTTTCTCGGCATTGC R: TAGCGACAGCGGTAGGACAGC
LSS	F: GAGGACCCGCTGGTCCA R: CCACACTGTTCCTGTGCGC
NSDHL	F: TTTGTGATCGGGAACGGGAAGAAC R: TTCGTCATTGGTGATGTGGAAGGC
DHCR24	F: CCTCTTCCTCCTGCCGCTCTC R: TGCCCTGCTCCTTCCATTCCC
RORC	F: CAATGGAAGTGGTGCTGGTCAGG R: GGGAGCGGGAGAAGTCAAAGATG
GAPDH	F: ACATCATCCCTGCTTCTACTGG R: CTCGGACGCCTGCTTCAC