Primer | Sequence (5′–3′) | Band Size |
---|---|---|
GR10 | GCATTGCCCCTAGGACCAGAGTC | 90 |
GR11 | CAAGCTGACACATCTGCCCGGAG | |
GR114 | GCAGGACCCCTGCAGG | 365 |
GR11 | CAAGCTGACACATCTGCCCGGAG | |
GR10 | GCATTGCCCCTAGGACCAGAGTC | 224 |
GR84 | TCGCTGTCTGAGCGGTACAGGAAG | |
IM1 | TCCGGGCAGATCTGTCAGCTT | 313 |
IM2 | ACTGGGAACGGGCACATTGGT | |
MS-FB11 | CCGGGCAGATCTGTCAG | 247 |
MS-FB12 | CCTGAGCAAATGGTGTTACG |
The positions of the primers are shown schematically in Fig. 1. For the GR primer set, GR29 (CAGGGTGRTCCACATCGTGG) was used to prime PDE4A specific first-strand cDNA synthesis. For the others (IM and MS-FB sets), the PDE4A-specific primer used was ESH4 (TGGTGATTCTCGAGCACCGAC). GR10/GR84 are used in the same way as IM1 and IM2. Both primer pairs were validated against positive and negative controls and amplification products were cloned and sequenced to confirm specificity. The IM1/IM2 pair used PCR conditions of 95°C for 5 min, 40 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, 72°C for 10 min, and then 4°C before use. The MS-FB11/MS-FB12 pair utilized PCR conditions of 35 cycles; 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min.