Apoptotic Gene Expression Analysis

Analyses of mRNA transcripts of key pro- and anti-apoptotic marker genes were conducted with MCF-7 cells exposed to 100, 400 or 800 µg/mL doses of F3 for 72 h [16 (link)]. Briefly, RNA was isolated by use of the Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the instructions of the manufacturer. Assessments of RNA quality, genomic DNA elimination and cDNA synthesis were carried out as previously described [16 (link)]. Amplification programs and PCR amplicon specificity were performed and assessed by use of a Rotor-Gene Q 5-Plex HRM thermal cycler (Qiagen, Germany) with QuantiTect SYBR-Green PCR Kit (Qiagen, Germany) as previously documented following standard protocols [16 (link)]. The following primers were used: Hs_P53_1_SG QuantiTect Primer Assay (QT00060235); Hs_CASP3_1_SG QuantiTect Primer Assay (QT00023947); B-cell lymphoma 2 Hs_BCL2_1_SG QuantiTect Primer Assay (QT00025011); Bcl-2-like protein Hs_BAX_1_SG QuantiTect Primer Assay (QT00031192); and 18S rDNA house-keeping (HK) gene Hs_RRN18S_1_SG QuantiTect Primer Assay (QT00199367). PCR thermal cycling program and gene expression analysis to determine the fold-change relative to the 18S gene were essentially performed as previously reported [16 (link)].