Tometer (Wilmington DE, USA) and stored at -80 before use. Complimentary DNA (cDNA) was synthesized using SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, USA). A total of 20 l of volume reaction which consisted of 10 l of 2X RT Reaction Mix, 2 l of RT Enzyme, 5 l of total RNA and 3 l ofTable 1 List of primers for qPCR analysisTarget mRNA GAPDH NF-B VCAM-1 ICAM-1 E-selectin Nox4 SOD1 CAT GPx Primer sequence F:tccctgagctgaacgggaag R:ggaggagtgggtgtcgctgt F: agtgcagaggaaacgtcagaa R: cattttaccacttggcaggaa F: agttgaaggatgcgggagtat R: ggatgcaaaatagagcacgag F: cagtcacctatggcaacgact R: ctctggcttcgtcagaatcac F: gtttggtgaggtgtgctcatt R: cattttaccacttggcaggaa F: cagaaggttccaagcaggag R: gttgagggcattcaccagat F: tggccgatgtgtctattgaa R: cacctttgcccaagtcatct F: gccattgccacaggaaagta R: ccttggtgagatcgaatgga F: ccaagctcatcacctggtct R: tcgatgtcaatggtctggaaDEPC-treated water was incubated at 25 for 10 minutes for primer annealing then at 50 for 30 minutes for reverse transcription. Following this, the reaction was terminated at 85 for 5 minutes, chilled on ice for 1 minute and 1 l of E. coli RNase H was added to the mixture. The cDNA was further incubated at 37 for 20 minutes and stored at -20 until use. Subsequently qPCR was carried out to determine the mRNA expression of NF-B, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx. Glycerylaldehyde-3-phosphate MK-1439 supplier dehydrogenase (GAPDH) was used as the reference gene. Primer 3 software [22] was used to design the primers from NIH GenBank database. The primer sequences for NF-B, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx are as listed in Table 1. The qPCR reaction was performed with 1 l of cDNA, 5 M of each forward and reverse primer and 12.5 l of IQ SYBR Green Supermix (Bio-Rad, USA) in BioRad iCycler (Bio-Rad, USA) with reaction profile of: 40 cycles of 95 (10 seconds) and 61 (30 seconds). The reaction kinetic of each primer set and protocol was verified with melting profile and product size was further confirmed with 2 agarose gel electrophoresis stained with ethidium bromide (Sigma, St Louis, USA). The threshold cycle (C T ) value was determined and the relative mRNA expression of the genes was calculated as follows: 2CT with CT = CT GAPDH – CT gene of interest.Statistical analysisData was tested for normality using Kolmogorov-Smirnov test and all variables were normally distributed. Data was expressed as means of fold change ?SEM. Statistical analysis between two groups was performedGenbank accession no NM_002046 NM_003998 NM_001078 NM_000201 NM_000450 NM_016931 NM_000454 NM_001752 NM_PCR product size (bp) 217 163 143 179 163 129 108 103Ugusman et al. BMC Complementary and Alternative Medicine 2011, 11:31 http://www.biomedcentral.com/1472-6882/11/Page 4 ofusing paired t-test in SPSS version 16.0 software. Values of p < 0.05 were considered statistically significant.ResultsEffects of AEPS on NF-B mRNA expression in HUVECsBoth AEPS and H 2 O 2 did not show any significant changes in the mRNA expression of NF-B (Figure 2).Effects of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26266977 AEPS on VCAM-1, ICAM-1 and E-selectin mRNA expression in HUVECshighest increase was observed in the CAT expression (2.2-fold) followed by GPx (1.3 fold) and SOD1 (1.2fold). In the oxidative stress-induced group, HUVECs treated with H2O2 also showed significantly higher level of SOD1, CAT and GPx mRNA expressions compared with the control group. HUVECs treated with both AEPS and H2O2 had significantly higher level of SOD1, CAT and GPx mRNA expre.