Osphate dehydrogenase (GAPDH) mRNA in the cells of each treatment group
Osphate dehydrogenase (GAPDH) mRNA in the cells of each treatment group was assessed by real-time quantitative PCR. Moreover, five transcripts related to the cell - including tissue inhibitor of metallopeptidase-3 (TIMP-3); hyaluronan synthase 1 (HAS-1); HAS-2; aggrecan (AGG); and collagen, type 2, alpha 1 chain (COL2A1) - were also quantified in all four groups to check the specificity of mRNA suppression by the siRNA. An ABI Prism?7000 apparatus (Applied Bio-Page 3 of(page number not for citation purposes)Journal of Orthopaedic Surgery and Research 2009, 4:http://www.josr-online.com/content/4/1/systems; Foster City CA, USA) was used to perform the quantitative analysis using SYBR?Green JumpStartTM Taq ReadyMixTM (Sigma) incorporation for dsDNA-specific fluorescent detection dye. Quantitative analyses of cell MMP3, TIMP-3, HAS-1, HAS-2, AGG and COL2A1 cDNA were performed in comparison with GAPDH as an endogenous control [16,17], and were run in separate wells. PCR was performed by using 2 l of each sample of cDNA and specific amplification primers. The primer sequences were designed for PCR amplification according to the human cDNA sequence (Table 1) using Primer Express?Software v2.0 (Applied Biosystems). Standard curves were generated for both target and endogenous control genes using serial dilution of plasmid DNA (101 - 108 molecules). The PCRs were performed in 20 l reaction volume containing 10.2 l SYBR?Green universal master mix (Sigma), optimal levels of forward and reverse primers, and 2 l of embryonic cDNA. During each PCR, reaction samples from the same cDNA source were run in duplicate to control the reproducibility of the results. A universal thermal cycling parameter (an initial denaturation step at 95 for 10 min, and 45 cycles of denaturation at 95 for 15 s and 60 for 60 s) was used PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14985054 to quantify each gene of interest. After the end of the last cycle, a dissociation curve was generated by starting the fluorescence acquisition at 60 and taking measurements at 7 sec intervals until the temperature reached 95 . Final quantitative analysis was done using the relative standard curve method, as used in Nganvongpanit et al. (2006) [16,17]. Results are reported as the relative expression level compared to the calibrator cDNA after normalization of the transcript amount to the endogenous control.Statistical analysis Results of cells morphology and proteoglycans were displayed as mean ?SD. The mRNA expression analysis for studied genes in all treatment groups was based on the relTable 1: Set of primers used for real-time quantitative PCRative standard curve method. All data were analyzed using the Statistical Analysis System (SAS) version 8.0 (SAS Institute, Inc.; Cary NC, USA) software package. Differences in mean values between two or more experimental groups or developmental stages were tested using ANOVA followed by multiple pairwise comparisons using a t-test. Differences of p < 0.05 were considered to be significant.ResultsEffect PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/19372634 of IL-1 treatment Treatment of cells using 10 ng/ml IL-1 for 24 h had an effect (p < 0.05) on cell morphology, proteoglycan production and gene expression (Table 2).Viability and mitotic rates in IL-1 treated groups were decreased compared to the non-treated groups (p < 0.05). But the apoptosis rate in IL-1 Cyclosporin D treated groups was increased (p < 0.05). The level of proteoglycan production (HA and GAG) decreased compared to groups not treated with IL-1 (p < 0.05). The relative expression of MMP-3 in IL-.
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