334 Table 1 Primer sequences for the selected genes used for validation of the microarray data using semi-quantitative RT–PCR and real-time RT–PCR Genes Nutr (2010) 5:331–341 Gene Primer sequence Glyceraldehyde-3-phosphate dehydrogenase (GADPH) (NM_002046) Phosphodiesterase 3A (PDE3A) (NM_000921) Interferon-induced protein with tetratricopeptide repeats (IFIT1) (NM_001548) Glutathione S-transferase A1 (GSTA1) (NM_145740) Microsomal triglyceride transfer protein (MTTP) (NM_000253) Forward: 50-TGCCTCCTGCACCACCAACTGC-30 Reverse: 50-AATGCCAGCCCCAGCGTCAAAG-30 Forward: 50-GAACAGATGACACTGCTCAAGTT-30 Reverse: 50-GAGCAAGAATTGGTTTGTCCAG-30 Forward: 50-AGAACGGCTGCCTAATTTACAG-30 Reverse: 50-GCTCCAGACTATCCTTGACCTG-30 Forward: 50-AGCCGGGCTGACATTCATCT-30 Reverse: 50-TGGCCTCCATGACTGCGTTA-30 Forward: 50-CGTTCGGCATCTACTTACAGC-30 Reverse: 50-GTTCTCCTCCCCCTCGTCAG-30 Product size (bp) 457 180 73 230 180 cDNA and primer pairs for the selected up-regulated and down-regulated genes as well as a housekeeping gene, GADPH, as listed in Table 1 were used. The PCR ampli- ﬁcation was performed in 0.2 ml MicroAmp Optical 8-tube strips in a ﬁnal volume of 20 ll containing a mix- ture of cDNA (30 ng), reverse and forward primers (1 lM), pre-prepared Power SYBR Green PCR master mix containing SYBR Green 1 dye, AmpliTaq Gold DNA Polymerase dNTPs, dUTP, Passive Reference 1 and optimized buffer components. The PCR parameters con- sisted of 40 cycles of ampliﬁcation with initial denaturation at 95 C for 15 s, annealing of primers and elongation of the newly synthesized strands at 60 C for 60 s. The PCR mixture was initially held for 10 min at 95 C for AmpliTaq Gold DNA polymerase activation. The comparative CT method (DDCT) was chosen for the relative quantitation of gene expression. Each sample type was run in triplicate. mRNA levels of the selected genes were normalized against GADPH mRNA. Results MTT cell proliferation assay The viability of HepG2 cells (%) in response to different concentrations of the methanol extracts of the T. indica fruit pulp is presented as a log plot in Fig. 1a. The plot shows a concentration-dependent decrease in cell viability with increasing concentrations of the methanol extracts of the T. indica fruit pulp. Cell viability was maintained above 80% even at a concentration of 1,000 lg/ml. The IC50 was found to be 4.57 mg/ml. Figure 1b shows the pictures of the cells viewed under light microscope, when the cells were treated with 300 lg/ml and 4.57 mg/ml of the methanolic extracts. In this study, we investigated the 123 effects of methanol extracts of the T. indica fruit pulp on gene expression patterns in cultured human HepG2 cells at a concentration of 300 lg/ml where cell viability was still above 90%. Normalization and visualization of microarray data Microarray data were initially visualized using a principal components analysis (PCA) plot (Fig. 2). The data from the control group (n = 3) were shown in blue, whereas those in the treated group (n = 3) were indicated in red. Each ball represents gene expression data generated from a sample that was applied on a gene chip. Figure 2a shows the data that were generated from the three biological replicates. From the plot, sample outliers were not detected both in the control and in treated group. The control group is also distinguishably separated from those of the treated samples. Each biological replicate comprising a control and a treated sample was also analysed on a different date (scan date), and the results are illustrated in Fig. 2b. It clearly shows that when analysed together, the control and the sample in each replicate are always grouped together. Figure 3 shows hierarchical clustering of genes in HepG2 cells in response to treatment with methanol extracts of the T. indica fruit pulp. Each lane represents an array. Up-regulated genes in treated samples were shown in orange, and down-regulated genes were shown in green. The pattern of the gene expression was similar in the biological replicates. In response to fruit pulp extracts of 300 lg/ml, 827 genes were signiﬁcantly down-regulated, and a further 674 genes were signiﬁcantly up-regulated. Amongst the signiﬁcantly up-regulated genes were MT1M, MT1F, MT1X, GSTA1, GSTA2 and GST02 (Table 2). In addition, APOA4, APOA5 and ABCG5 were also up-regu- lated. Genes that were signiﬁcantly down-regulated include PDE3A, PTTG1, CYP1A1, IFIT1 and MTTP (Table 2).