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Table 2 Primers, product length and specific amplification conditions for qPCR

From: In vitromodel for the analysis of synovial fibroblast-mediated degradation of intact cartilage

Gene Primer forward Primer reverse Accession number T annealing Melting T product
Human/bovine Aldolase A 5'-TCATCCTCTTCCATGAGACACTCTA-3' 5'ATTCTGCTGGCAGATACTGGCATAA-3' [GenBank: NM_000034] 58°C 88°C
Human MMP-1 5'-GACCTGGAGGAAATCTTGC-3' 5'-GTTAGCTTACTGTCACACGC-3' [GenBank: NM_002421] 58°C 86°C
Human MMP-3 5'-CTCACAGACCTGACTCGGTT-3' 5'-CACGCCTGAAGGAAGAGATG-3' [GenBank: NM_002422] 58°C 81°C
Human IL-6 5'-ATGAACTCCTTCTCCACAAGCG-3' 5'-CTCCTTTCTCAGGGCTGAG-3' [GenBank: NM_000600] 60°C 86°C
Human IL-8 5'-GCCAAGAGAATATCCGAACT-3' 5'-AGGCACAGTGGAACAAGGACTTGT-3' [GenBank: NM_000584] 60°C 78°C
Bovine MMP-1 5'-CAAGAGCAGATGTGGACCAA-3' 5'-CTGGTTGAAAAGCATGAGCA-3' [GenBank: NM_174112] 61°C 83°C
Bovine MMP-3 5'-CTGGTGTCCAGAAGGTGGAT-3' 5'-TAGGCGCCCTTGAAGAAGTA-3' [GenBank: AB043995] 61°C 83°C
Bovine IL-6 5'-ATGAACTCCCGCTTCACAAG-3' 5'-CCTTGCTGCTTTCACACTCA-3' [GenBank: NM_173923] 61°C 83°C
Bovine IL-8 5'-TGCTCTCTGCAGCTCTGTGT-3' 5'-CAGACCTCGTTTCCATTGGT-3' [GenBank: NM_173925] 64°C 81°C
Bovine Col II (α 1 chain) 5'-CATCTGGTTTGGAGAAACCATC-3' 5'-GCCCAGTTCAGGTCTCTTAG-3' [GenBank: NM_001001135] 61°C 83°C
Bovine COMP 5'-ATGCGGACAAGGTGGTAGAC-3' 5'-TCTCCATACCCTGGTTGAGC-3' [GenBank: X74326] 61°C 87°C
  1. General amplification protocol (40 cycles): initial denaturation for two minutes at 95°C; denaturation for 15 seconds at 95°C, specific primer annealing temperature (see table) for 15 seconds, amplification at 68°C for 20 seconds, additional heating step to 5°C below the melting temperature of the PCR product (see table). General melting curve protocol (one cycle): denaturation for one second at 95°C; cooling to 5°C above the primer annealing temperature (holding for 10 seconds); heating to 95°C (0.1°C/second); final cooling for five minutes at 40°C.
  2. Col = collagen; COMP = cartilage oligomeric matrix protein; IL = interleukin; MMP = matrix metalloproteinase; qPCR = quantitative polymerase chain reaction.