Real-Time Reverse Transcriptase Quantitative PCR

To determine the chondrocyte-specific gene expression, the magnitude of mRNA was analyzed with RT-qPCR for collagen type II (COL2A1), collagen type I (COL1A1), aggrecan (ACAN), SOX9, SOX5, SOX6, matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 13 (MMP13), from chondrocytes cultivated in MACT (CaReS) grafts and OA chondrocytes in collagen type I hydrogels. Probes and primers were selected with the Universal Probe Library System (Roche) and by performing in silico PCR. Optimum conditions and annealing temperature were determined experimentally. cDNA was amplified with the FastStart TaqMan Probe Master (Roche Diagnostics GmbH) with gene-specific primers (Eurofins MWG Synthesis GmbH, Ebersberg, Germany) in triplicates using the iCycler iQ (Bio-Rad Laboratories, Hercules, CA). Target cycle threshold (CT) values were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and compared to the corresponding sample using the comparative C_T (ΔΔC_T) method without efficiency correction (see Table 1 ).

Sequences of Primers and Conditions Used in Reverse Transcriptase Quantitative Polymerase Chain Reaction.