Table 2.
Sequences of PCR primers
| Group | Gene | Primer sequence forward 5′–3′ (exon) | Primer sequence reverse 5′–′3′ (exon) | Product size | Acc. number |
|---|---|---|---|---|---|
| Collagens | COL1A1 | GTCTTCCTGGCCCCTCTGGTG (40) | TCGCCCTGTTCGCCTGTCTCA (45) | 390 | NM_000088 |
| COL3A1 | CAGGGGCCCCAGGACTTAGAG (30) | GGGCCAGGAGGACCAATAGGT (33) | 249 | NM_000090 | |
| COL4A1 | CAATGCCCTTCCTGTTCTGC (51) | GTGGACGGCGTAGGCTTCTT (53∼54) | 451 | NM_001845 | |
| MMPs | MMP2 | ACATCAAGGGCATTCAGGAG (8) | TGAACCGGTCCTTGAAGAAG (9∼10) | 168 | NM_004530 |
| MMP9 | GGGAAGATGCTGCTGTTCA (11) | TCAACTCACTCCGGGAACTC (13) | 202 | NM_004994 | |
| MMP14 | GCAGAAGTTTTACGGCTTGC (2) | CTTGGGGGTGTAATTCTGGA (4) | 199 | NM_004995 | |
| TACE/ADAM-17 | TCATTGACCAGCTGAGCATC (17) | CGCAGGAAAGGGTTTGATAA (20) | 233 | NM_003183 | |
| TIMPs | TIMP1 | CTGCGGATACTTCCACAGGT (3) | GTTTGCAGGGGATGGATAAA (5) | 209 | NM_003254 |
| TIMP2 | GATGCACATCACCCTCTGTG (4) | GTGCCCGTTCATGTTCTTCT (5) | 196 | NM_003255 | |
| TIMP3 | CTGACAGGTCGCGTCTATGA (3∼4) | AGTCACAAAGCAAGGCAGGT (5) | 165 | NM_000362 | |
| TIMP4 | CTTGGTGCAGAGGGAAAGTC (4) | GGCTGAACGATGTTCAACAAA (5) | 249 | NM_003256 | |
| Cytokines and receptors | TNFA | CAGAGGGCCTGTACCTCATC (2) | GGAAGACCCCTCCCAGATAG (4) | 219 | NM_000594 |
| TNFSFR1A | GTGCCTACCCCAGATTGAGA (7) | TGTCGATTTCCCACAAACAA (8∼9) | 175 | NM_001065 | |
| TNFSFR1B | GGAAACTCAAGCCTGCACTC (4) | TGCAAATATCCGTGGATGAA (5) | 224 | NM_001066 | |
| IL-6 | CCACACAGACAGCCACTCAC (2) | TTTCACCAGGCAAGTCTCCT (3) | 217 | NM_000600 | |
| Ribosomal | RPL19 | CAATGCCAACTCCCGTCAGC (1∼2) | CTTGGTCTCTTCCTCCTTGG (6) | 487 | NM_000981 |
Primers were generated with the Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and were mRNA/cDNA-specific. Preliminary experiments were conducted to ensure that the number of cycles represented a linear portion for the PCR optical density (OD) curve. The specificity of each primer pair was verified by sequencing the amplified products. PCR cycles were as follows: 25 (TIMP2 aponeuroses), 27 (MMP2 and TIMP3), 28 (COL1A1 aponeuroses), 29 (RPL19), 30 (TIMP1 and COL4A1; and TIMP2 and TIMP3 muscles), 31 (TNFSFR1B and COL3A1; and COL1A1 muscles), 32 (MMP2 muscles), 35 (MMP14, TIMP4, TNFA and TNFSFR1A; and IL-6 aponeuroses), 36 (IL-6 muscles), 37 (MMP9 aponeuroses) or 40 (MMP9 muscles).
COL1A1, collagen, type I, alpha-1;
COL3A1, collagen, type III, alpha-1;
COL4A1, collagen, type IV, alpha 1.