Total RNA was extracted, and reverse transcription was performed with 460 ng of total RNA in a total volume of 10 µl under conditions of 37 °C for 15 min, 85 °C for 5 s and 4 °C for 5 min. cDNA was synthesized using the Reverse Transcriptase kit according to the manufacturer’s protocol (PrimeScript RT Master Mix, RR036A, Takara, Biomedical Technology Co., Ltd.), and the cDNA product was then amplified by real-time PCR in a total volume of 20 µl according to the manufacturer’s protocol (SYBR premix Ex Taq™ II, RR820A, Takara, Biomedical Technology Co., Ltd) using gene-specific primers (Table 1 ) on an ABI 7300/7500 real-time PCR instrument (Applied Biosystems, Carlsbad, CA, USA). To 2 μl of cDNA, 0.8 μl of primers for the gene of interest and 0.8 μl of primers for the reference gene were added, and the reaction conditions were 40 cycles of 95 °C for 30 s, 95 °C for 5 s, and 55 °C–60 °C for 34 s. Relative mRNA expression levels were calculated using the ΔΔCq method and normalized to β-actin mRNA levels. Individual samples were assayed in triplicate, and the average quantification cycle (Cq) was calculated for the gene of interest and the reference gene. Based on the difference between both Cq values, the comparison was calculated. All primers were synthesized by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China). The primer efficiencies were 99.98%-101.01%.
Primer used for qPCR
| Gene | Accession number | Nucleotide sequence (from 5′ to 3′) | Amplicon (bp) |
|---|---|---|---|
| CYP-7A1 | NM-012942.2 | F:TGGCTGTCATGTGCAGGCGCCAA R:ACGCACTGCCTCAGCAAACACACA | 159 |
| CYP7B1 | NM_019138.1 | F: ACCACAGTCGCATGTTTCTGGGCA R: TCCGCTAAGCTTCTCTGCCACCCT | 108 |
| TGR5 | NM_177936.1 | F:GCCCGCTGTGGGGGCCACTGCCCT R:GGGTGCATCACGGCACACCGCCCGC | 109 |
| β-actin | XM-032887061.1 | F:AAGTCCCTCACCCTCCCAAAAG R: AAGCAATGCTGTCACCTTCCC | 96 |
CYP7A1 Cholesterol 7α-hydroylase, CYP7B1 Microsomal oxysterol 7a-hydroxylase, TGR5 Takeda G-protein-coupled receptor-5
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