β actin, beta-2 microglobulin (B2M) and 18S were selected from a panel of reference genes using the geNorm component of the qBase 2.0 relative quantification model (Biogazelle, Belgium) as they were expressed stably across treatment groups (Control vs. MA) [36 (link)–38 (link)].
The mRNA expression of P-glycoprotein (P-gp) [36 (link)], CYP1A2, PXR, GR, 11βHSD1, 11βHSD2, MR, p50, p65, MCP1, SOD1, SOD2 and reference genes in liver samples were measured using KiCqStart SYBR Green qPCR ReadyMix Low Rox on a ViiA7 Fast Real-time PCR system (Applied Biosystems, California, USA) as previously described [35 (link), 36 (link)]. The reactions were quantified by setting the threshold within the exponential growth phase of the amplification curve and obtaining corresponding Ct values. DataAssist Software v3.0 (Applied Biosystems) [38 (link)] was used to find the 2−ΔCt, which shows the abundance of each transcript relative to the abundance of the three stable reference genes and is expressed as mean normalized expression. DataAssist 3.0 analysis software (Applied Biosystems, California, USA) was used to normalise the abundance of target genes relative to the abundance of reference genes and expressed as mRNA mean normalised expression (MNE) ± SEM.
The mRNA expression of P-glycoprotein (P-gp) [36 (link)], CYP1A2, PXR, GR, 11βHSD1, 11βHSD2, MR, p50, p65, MCP1, SOD1, SOD2 and reference genes in liver samples were measured using KiCqStart SYBR Green qPCR ReadyMix Low Rox on a ViiA7 Fast Real-time PCR system (Applied Biosystems, California, USA) as previously described [35 (link), 36 (link)]. The reactions were quantified by setting the threshold within the exponential growth phase of the amplification curve and obtaining corresponding Ct values. DataAssist Software v3.0 (Applied Biosystems) [38 (link)] was used to find the 2−ΔCt, which shows the abundance of each transcript relative to the abundance of the three stable reference genes and is expressed as mean normalized expression. DataAssist 3.0 analysis software (Applied Biosystems, California, USA) was used to normalise the abundance of target genes relative to the abundance of reference genes and expressed as mRNA mean normalised expression (MNE) ± SEM.
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