Table 1

Primers, product length and specific amplification conditions for qPCR.

Gene

Primer upstream(5'→3')

Primer downstream(3'→5')

Accession number

Product length in bp

T annealing

Melting T product


Aldolase

5´-TCATCCTCTTCCATGAGACACTCTA-3´

3´-ATTCTGCTGGCAGATACTGGCATAA-5´

NM_000034

314

58°C

88°C


Aggrecan

5´-CAGAGTTCAGTGGGACAGCA-3´

3´-AGACACCCAGCTCTCCTGAA-5´

NM_173981

189

60°C

84°C


Coll II

5´-CATCTGGTTTGGAGAAACCATC-3´

3´-GCCCAGTTCAGGTCTCTTAG-5´

NM_001001135

600

61°C

83°C


Coll I

5´-AGCCAGCAGATCGAGAACAT-3´

3´-ACACAGGTCTCACCGGTTTC-5´

NM_001034039

185

60°C

86°C


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 above) 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 above). 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. Col = collagen. qPCR = quantitative polymerase chain reaction.

Pretzel et al. Arthritis Research & Therapy 2013 15:R59   doi:10.1186/ar4231

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