Custom specific primers

RNA (50ng) harvested from three independent batches of synoviocytes, SFCs, Chondrocytes, two independent batches of CPCs was analyzed using Affymetrix Bovine Genome Arrays as previously described.^{17 (link)} Statistical fold change expression was applied via 1-way analysis of variance (ANOVA) model using Method of Moments.^{20 (link)} Fisher’s Least Significant Difference (LSD) was used as the contrast method. Hierarchical cluster analysis was generated using Partek Genomics Suite software. Quantitative PCR (qPCR) reactions were performed as previously described using custom specific primers purchased from Integrated DNA Technologies (Coralville, IA) (Table 1). All qPCR experiments were performed in triplicate. β-actin was used as a reference gene. Fold change was calculated by the 2^−ΔΔCt method.

CPCs were treated with SDF-1 for 24 hrs with or without 2 hr-pretreatment of 200 ng/ml AMD3100. To determine the effect of MAPK inhibitor on SDF-1 stimulated VEGF expression, CPCs were also treated with 50 ng/ml SDF-1 for 24 hrs with or without 1 hr-pretreatment of 10 μM MAPK inhibitor (SB203580, PD98059, SP600125). Total RNA was extracted with the combination for Trizol Reagent (Life Technologies) and RNeasy Mini Kit (QIAGEN, Valencia, CA), according to the manufacturer’s instructions. Real time PCR analysis was carried out using TaqMan reverse transcription reagents and SYBR green PCR Master Mix (Applied Biosystems). Custom specific primers were purchased from Integrated DNA Technologies (Coralville, IA) (Table 1). β-actin was used as an internal reference gene. Relative quantitative method (2^-ΔΔCT) was used to calculate the relative expression levels of VEGF among different groups.

50 ng RNA was reverse transcribed to complimentary DNA using TaqMan reverse transcription reagents (Applied Biosystem, Grand Island, NY). qPCR reactions were performed with SYBR Green reagents (Applied Biosystem, Grand Island, NY) and custom specific primers (Integrated DNA Technologies, Coralville, IA), including CD 68 (Forward: GGA GTA ATG GTT CCC AGC CC, Reverse: CTG CAG TGG ATC CTG CTT GA), CD 14 (Forward: ACC ACC CTC AGT CTC CGT AA, Reverse: GCC GAG ACT GGG ATT GTC AG), GULP1 (Forward: TGG ATG CAT ACT CCC GAA GC, Reverse: AGC TGG CAA TTG TGT TGA ACT), LAMP1 (Forward: GTG AAG AAT GGC AAC GGG AC, Reverse: TTA TTC TGG GGC CCA CTC CT). All qPCR experiments were performed in triplicate. Each gene expression level was normalized to β-actin. The fold change was calculated by the 2^−ΔΔCt method.