Effect of Serenoa repens (Permixon®) on the expression of inflammation-related genes: analysis in primary cell cultures of human prostate carcinoma

Table 2 Primers sequences and amplification conditions

Gene	Sequences 5’-3’	Cicli	Annealing (temp C)	Size (bp)
GADPH	ACATGTTCCAATATGATTCC	30	60°×30”	180
GADPH	TGGACTCCACGACGTACTCAG	30	60°×30”	180
Il-6	CCTCCAGAACAGTTTGAGA	30	56°×1’	280
Il-6	CCTTAAAGCTGCGCAGAATG	30	56°×1’	280
CCL-5	CTCGCTGTCATCCTCATTGCT	30	62°×30”	394
CCL-5	TACTCCCGAACCCATTTCTTCTC	30	62°×30”	394
CCL-2	ATG AAA GTC TCT GCC GCC CTT CTG T	36	60°×30”	286
CCL-2	AGT CTT CGG AGT TTG GGT TTG CTT G	36	60°×30”	286
COX-1	TGC CCA GCT CCT GGC CCG CCG CTT	30	60°×1’	304
COX-1	GTG CAT CAA CAC AGG CGC CTC TTC	30	60°×1’	304
COX-2	TTC AAA TGA GAT TGT GGG AAA AT	30	60°×1’	305
COX-2	AGA TCA TCT CTG CCT GAG TAT CTT	30	60°×1’	305
iNOS	TGG TGC TGT ATT TCC TTA CGA GGC GAA GAA GG	35	60°×45”	259
iNOS	GGT GCT TCT TGT TAG GAG GTC AAG TAA AGG GC	35	60°×45”	259

The PCR amplification programme was as follows: an initial period of 5 min at 95°C, followed by a variable number of cycles of denaturation at 95°C for 30 sec, annealing (see Table 1) and finally 30 sec of extension at 72°C. The programme was terminated with a period of 10 min at 72°C. To be within the exponential phase of the semiquantitative PCR reaction, the appropriate number of cycles was newly established for every set of samples. PCR products were separated by gel electrophoresis on 15% agarose gels and visualized by ethidium bromide staining. All gels were scanned and the normalizedintensities of all reverse transcription (RT)-PCR products were determined by the BioRad gel documentation system (BioRad, Hercules, CA, U.S.A.). Mean ± SEM intensities were calculated for all RT-PCR experiments.

ISSN: 1476-9255