Quantitative analysis of gene expression in cancer cells treated with plant extracts

Quantitative real time polymerase chain reaction (qRTPCR) was used to study the effect of Tritticum spelta and Salix mucronata plant extract on p53, BCL2, Cyclin D, MMP9 and VEGF gene expression. Total RNAs were extracted from the untreated and the treated HepG2 and Caco-2 cancer cell lines using Gene JET RNA Purification Kit (Thermo Scientific, USA). Then cDNA was synthesized utilizing cDNA Synthesis Kit (Thermo Scientific, USA). Real time PCR was performed using SYBR green master mix and specific primers which are shown in Table 1 in supplementary materials. The 2^−ΔΔCT equation was used to calculate the change in gene expressions in the treated cancer cells relative to untreated cancer cells. The expression of target genes was calculated using the comparative Ct method (threshold cycle number at cross-point between amplification plot and threshold). The CT values of each target gene were normalized to that of β-actin according to manufacturer’s instructions and the change in expression (2^−ΔΔCT) was calculated.