Real-time RT-PCR determination

Analysis of mRNA expression levels for collagen types I, III, TGF-β1 and ACE were performed with primers designed to detect rat gene products: Collagen type I used primers, FWD: 5′-CCTGCCGATGTCGCTATCC-3′, and REV: 5′-TTGCCTTCGCCCCTGAG-3′; Collagen type III used primers, FWD: 5′-GCCTCCCAGAACATTACATACC-3′, and REV: 5′-CTGTCTTGCTCCATTCACCAG -3′; TGF-β1 used primers, FWD: 5′-TGGCGTTACCTTGGTAACC-3′, and REV: 5′-GGTGTTGAGCCCTTTCCAG-3′; ACE used primers, FWD: 5′-ATGAGGCTATTGGAGATGTTTTG-3′, and REV: 5′-TCCTTGGTGATGCTTCCGT-3′; GAPDH used primers, FWD: 5′-TGGCATGGACTGTGGTCATG-3′, and REV: 5′-TGGGTGTGAACCACGAGAAA-3′.

Real-time RT-PCR and datum analysis were carried out with realplex 7500 (Applied Biosysems, USA).

All data obtained with collagen types I and III, TGF-β1, ACE primers were normalized to the GAPDH primers, namely the relative integrated intensity was taken.