PCR and RT-PCR primers were designed by using Fuzzy Detective Theory. The characteristic of fuzzy detective theory is to introduce the experience into fuzzy detection. The PCR, RT-PCR, and antisense primers were set to contain 42-62% of GC content. For the next steps, less homologous primers were chosen versus self-mRNA, GAPDH (glyceraldehyde 3-P-dehydrogenase), and beta-actin sequence. Designed PCR primers were used to analyze gene expression levels, and molar concentrations were estimated by quantitative competitive RT-PCR assays. Antisense (As-) oligonucleotides were designed to inhibit translation of target mRNA and to contain initiation cordon AUG region of mRNA, divide sequences from 15-19 bases upstream to downstream were scanned homology on its entire mRNA. For the control, the sense oligonucleotides were designed by the same procedure, and mismatched oligos were designed by each nucleotide shuffling. All the inhibitory effects of designed antisenses were confirmed by increased blood pressure of rat injecting As-nNOS to NTS (Nucleus Tractus Solitarii), decreased transmigration activity by As-MIP-lb using ATL cells, and profilin gene expression suppression.
