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Description of the Beacon designer sequences used to quantify gene expression and real-time reaction efficiencies of PCR assays |
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| Gene |
Accession number |
Sequences |
Product size (base pairs) |
Efficiency |
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| iNOS |
U14640 |
Probe: 5'-FAM-CGCGATCCCTGCTTGGTGGCGAAGATGAGCGATCGCG-DABCYL-3' Forward: 5'-GTAACAAAGGAGATAGAAACAACAGG-3' Reverse: 5'-CAGCTCCGGGCGTCAAAG-3' |
81 |
1.98 ± 0.06 |
| COX-2 |
AF031698 |
Probe: 5'-FAM-CGCGATCGTCAGAAATTCGGGTGTGGTACAGTTGATCGCG-DABCYL-3' Forward: 5'-CGAGGTGTATGTATGAGTGTAGG-3' Reverse: 5'-GTTGGGAGTGGGTTTCAGG-3' |
82 |
1.99 ± 0.03 |
| Aggrecan |
U76615 |
Probe: 5'-FAM-CGCGATCCACTCAGCGAGTTGTCAGGTTCTGAGATCGCG-DABCYL-3' Forward: 5'-TGGTGTTTGTGACTCTGAGG-3' Reverse: 5'-GATGAAGTAGCAGGGGATGG-3' |
79 |
1.97 ± 0.05 |
| Collagen type II |
X02420 |
Probe: 5'-FAM-CGCGATGCGTCAGGTCAGGTCAGCCATATCGCG-DABCYL-3' Forward: 5'-AAACCCGAACCCAGAACC-3' Reverse: 5'-AAGTCCGAACTGTGAGAGG-3' |
70 |
2.00 ± 0.05 |
| GAPDH |
U85042 |
Probe: 5'-HEX-CGCGATCCACCATCTTCCAGGAGCGAGATCCGATCGCG-DABCYL-3' Forward: 5'-TTCAACGGCACAGTCAAGG-3' Reverse: 5'-TTCAACGGCACAGTCAAGG-3' |
75 |
2.03 ± 0.01 |
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The Beacon Designer software was used to design forward and reverse primer and probe sequences for molecular beacon applications and were synthesized by Sigma Genosys Ltd, Cambridge, UK. Secondary structures were avoided using the Mfold programme and sequences were analyzed using Basic Local Alignment Search Tool to verify specificity. Probes contain fluorescein (FAM) or 6-carboxyhexafluorescein (HEX) as a 5'-reporter dye and 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) as 3'-quencher. Note that the arm sequences are underlined. COX, cyclo-oxygenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase. | ||||
Chowdhury et al. Arthritis Research & Therapy 2008 10:R35 doi:10.1186/ar2389 |
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