Genes Nutr (2010) 5:331–341 conventional Affymetrix eukaryotic RNA labelling proto- col (Affymetrix). Brieﬂy, the tcRNA (100 ng) isolated from the untreated (control) and the T. indica-treated HepG2 cells was ﬁrst converted to single-stranded sense strand DNA (cDNA) in two cycles using the whole tran- script (WT) cDNA synthesis, ampliﬁcation kit and sample clean-up module. In the ﬁrst cycle, the tcRNA was con- verted to double-stranded cDNA using random hexamers tagged with a T7 promoter sequence. Each strand of the double-stranded cDNA was then used as a template to synthesize antisense RNA (cRNA). In the second cycle, the cRNA was reversed transcribed into sense strand DNA in the presence of random hexamers (3 lg/ml) and dNTPs mix containing dUTP (10 mM). Sense strand DNA labelling and hybridization to the Affymetrix Human Gene 1.0 S.T arrays The sense strand DNA was cleaned up using the sample clean-up kit and then cleaved into small fragments using a mixture of UDP and apurinic/apyrimidinic endonuclease 1 or APE1. The fragmented sense strand DNA was then end-labelled through a terminal transferase reaction that incorporates biotinylated dideoxynucleotides using the WT terminal labelling kit. The fragmented, biotinylated sense strand DNA (5.5 lg) was then hybridized to the Affymetrix Human Gene 1.0 S.T array at 45 C for 16 h in a hybridization Oven 640. After hybridization, the arrays were stained and then washed in the Affymetrix Fluidics Station 450 under standard conditions. The stained arrays were then scanned at 532 nm using an Affymetrix GeneChip Scanner 3000, and CEL ﬁles for each array were generated using the Affymetrix Gene- Chip Operating Software (GCOS). The data were sub- jected to normalization, background correction and data summarization using Affymetrix Expression Console software. Microarray data normalization and analysis The CEL ﬁles were then converted to text ﬁles and exported to Partek Genomic Suite software to get the whole list of up-regulated and down-regulated genes. The whole gene lists generated were then normalized by ﬁltering out probeset IDs that did not have any annotation in the Partek software, using the Netaffx Analysis Center software. The gene sets were then subjected to a one-way analysis of variance (ANOVA) in the Partek Genomic software to determine signiﬁcantly expressed sets of genes, which was set according to P value less than 0.05 (P \ 0.05). Sig- niﬁcantly expressed genes were then re-ﬁltered to include only those with fold change difference of equal to or greater than 1.5. 333 Biological interpretation: gene ontology (GO) Additional information on the biological functions of the genes (genes products) that were signiﬁcantly regulated (P \ 0.05) with a fold change which was equal to or greater than 1.5 was determined using the Gene Ontology Enrichment tool in the Partek Genomic Suite Software. Validation of the microarray data using semi-quantitative reverse transcription polymerase chain reaction (RT–PCR) The microarray data were validated using semi-quantitative reverse transcription PCR (RT–PCR) as follows: tcRNA (2 lg) from control and T. indica-treated cells were reverse-transcribed to cDNA in a ﬁnal volume of 20 ll containing a mixture of ﬁrst strand buffer (1X), random hexamers (25 ng/ll), dNTP mix (0.5 mM), dithiothreitol (DTT) (0.1 M) and superscript II reverse transcriptase (10 units). The mixture was then incubated at 42 C for 50 min followed by inactivation at 70 C for 15 min. The cDNA was then used as templates for PCR ampliﬁcation of selected up-regulated and down-regulated genes using primer pairs listed in Table 1. A PCR mixture containing cDNA (2000 ng), PCR buffer (1 X), 50 and 30 primers for GADPH and the selected genes of interest (1 lM), dNTPs (10 mM), DMSO (10% v/v) and Taq DNA polymerase (5 units) were prepared to a ﬁnal volume of 50 ll with sterile distilled water (Sigma). PCR ampliﬁcation was carried out in a thermal cycler (Biometra) with initial DNA denaturation at 95 C for 3 min followed by 35 cycles of denaturation at 94 C for 5 s, annealing of primers to the cDNA templates at 55 C for 1 min and DNA extension/ elongation at 72 C for 3 min. The ﬁnal DNA extension was done at 72 C for 10 min. The PCR products were then separated through electrophoresis on a 2% agarose gel stained with 2 lg/ml ethidium bromide. The separated bands were visualized using a UV transilluminator, and the images were captured using a Canon CCD camera (BioRad Gel Doc 1000/2000). The intensity of the bands that corresponded to the amount of the PCR products of the selected regulated genes was then quantitated using Quantity One software (BioRad). Scanned values were expressed in arbitrary unit, and the expression of the selected genes relative to that of a housekeeping gene GADPH was then determined. Validation of the microarray data using real-time RT–PCR Validation of the microarray data using quantitative real- time PCR (qRT–PCR) was carried out in a StepOneTM Real-Time PCR System (Applied BioSystem). The same 123