Altered Interleukin-1 Receptor Antagonist and Interleukin-18 mRNA Expression in Myocardial Tissues of Patients with Dilatated Cardiomyopathy

Table 2 Primers used for RT-PCR

	Primer sequence (5′–3′)	PCR product size, bp	Protocol^a	Primer annealing, °C	PCR cycles
IL-1a	ATG-GCC-AAA-GTT-CCA-GAC-ATG TAC-GCC-TGG-TTT-TCC-AGT-ATC-TG	817	I	56	30
IL-1β	GAG-GAT-GAC-TTG-TTC-TTT-GAA-G GTT-GCT-CAT-CAG-AAT-GTG-GGA-G	1096	I	50	29
IL-lra	ATG-GCT-TTA-GAG-ACG-ATC-TGC AGT-AGA-ATT-TGG-TGA-CCA-TGA-C	465	III	56	23
IL-18	ATA-TGG-ATC-CAT-GGC-TGC-TGA-ACC-AGT-AG ATA-TCT-GCA-GCT-AGT-CTT-CGT-TTT-GAA-CAG	604	II	60	30
IL-l-RI	ATG-AGA-CAA-TGG-AAG-TAG-AC TAG-ATG-AAA-ACA-GAA-CAC-AC	372	III	56	21
Caspase-1	ATA-TGG-ATC-CGA-CAA-CCC-AGC-TAT-GCC-C ATA-TCT-GCA-GAT-GTC-CTG-GGA-AGA-GGT-A	880	III	56	22
Caspase-3	ATA-AAG-GGA-TCC-ATG-GAG-AAC-ACT-GAA-AAC-TCA-G AAC-CTG-CAG-TTA-GTG-ATA-AAA-ATA-GAG-TTC-TTT-TGT-GAG-C	857	II	64	30
18S rRNA	GTT-GGT-GGA-GCG-ATT-TGT-CTG-G AGG-GCA-GGG-ACT-TAA-TCA-ACG-C	348	IV	60	21
β-actin	GTC-AGA-AGG-ATT-CCT-ATG-TGG GGA-GCA-ATG-ATC-TTG-ATC-TTC	855	I	50	28

^aAfter the initial denaturation step (94°C; 2.5 min) 1 of the 4 following RT-PCR protocols was performed (D, denaturation; A, annealing; E, elongation). I: A (30 s, 94°C; 30 s at primer-specific annealing temperature); E (90 s, 72°C). II: same as I except annealing time 90 s. III (touch down PCR): after the denaturation step (30 s, 94°C) in the first cycle, A and E were 60 s at 66°C; A and E were reduced 1°C each following cycle until they reached 57°C; followed by cycles: D (30 s, 94°C); A (30 s, 56°C); E (90 s, 72°C). IV: D (30 s, 94°C); A (30 s, 60°C); E (30 s, 72°C).

ISSN: 1528-3658