Microarray and Quantitative real-time RT-PCT (qRT-PCR)

RNA was extracted from primary MPCs using RNeasy Mini Kit (Qiagen, Valencia, CA). Isolated 50 ng RNA was converted to SPIA amplified cDNA and biotinylated cDNA was placed onto Bovine Genome Arrays (Affymetrix Inc., San Carlos, CA). Arrays were scanned with the Affymetrix Model 3000 and data were collected using the GeneChip^® operating software (MAS) v5.0. Statistical analysis of the data (one-way ANOVA) and a heatmap & dendrogram were generated using Partek Genomics Suite software (Partek, St. Louis, MO)

qRT-PCR was performed with the SuperScript™ III Platinum^® SYBR^® Green One-Step qRT-PCR kit (Invitrogen) as described in previous study.²⁵ The following primer sequences were used: for GAPDH (as a reference gene), forward CAT GGG TTT AGC CAA CTG TG and reverse GAC GGA TGC TAC CTA CGG AT; for CHAD, forward GGG CCT CAA GCA ACT CAT CT and reverse CAG CTC CCG GAT CTT GTT GT; for COL1A2, forward CTG TTG GTA ACC CTG GTC CC and reverse CAC CCT TAG CAC CAG TGA GG; for COL2A1, forward TGA AGA CAC CAA GGA CTG CC and reverse GCA GTG GCG AGG TCA GTA G; for IL8, forward CAA CCT TCC TGC TGT CTC TG and reverse GGT CCA CTC TCA ATC ACT CT; for MMP9, forward CCC GGA TCA AGG ATA CAG CC and reverse GGG CGA GGA CCA TAC AGA TG.