7) and triplicate assessment by using microplate (BioTeck, USA). First-strand cDNA was synthesized using 1 μg of total RNA by reverse-transcription using iScript™ cDNA synthesis kit (Bio-rad, California) as instructed by the manufacturer. For real-time PCR analysis of MMP1 and
GAPDH gene expression was carried out using iQ™ http://www.selleckchem.com/products/chir-99021-ct99021-hcl.html SYBR® Green Supermix (Bio-rad, California), the primers used were: • MMP1 forward: AGTCAAGTTTGTGGCTTATGGA Briefly, the reaction mixture containing 2 μL cDNA, 1 μL forward primer (0.5 μM), 1 μL reverse primer (0.5 μM), 10 μL iQ SYBR Green Supermix, and 6 μL RNase-free water was prepared. The real-time PCR program was set as follows: initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, and then 61 °C for 30 s. Finally, the melting curve program was performed at the end of each reaction. The relative levels of mRNA expression was assessed
by the comparative Ct method (DDCT method), which normalize the mRNA level of negative control to that of reference gene GAPDH. To construct MMP1 target reporter plasmid, as shown in Fig. 1, the MMP1 cDNA (sequence 150–953) fused with Kozak sequence  and  at 5′-end to initiate translation process and incorporated 2 restriction sites to facilitate subcloning reaction was first amplified (831 bp fragment) Sotrastaurin supplier by PCR (Fig. 2) and subcloned into pAcGFP1-N3 vector, using HindIII Non-specific serine/threonine protein kinase and BamHI cutting sites, downstream the immediate early promoter of CMV (PCMV IE) and followed in frame by the green fluorescent protein AcGFP1 coding sequences. Although the partial MMP1-AcGFP1 fusion DNA could be transcribed under control of CMV promoter, and translated by Kozak sequence, the fluorescent intensity was not satisfied (data not shown). It might be because the molecular of N-terminal fused MMP1 partial protein was too large, which consequently affected the green fluorescent protein folding or its function. To overcome this issue, three potent siRNA target DNAs, 506-MMP1, 859-MMP1 and 891-MMP1 as shown in Fig. 1B–D, were constructed individually
to pAcGFP1-N3 plasmid. Since the length of target gene was about 25–26 bp, forward and reward oligonucleotides were annealed by cooling down from 95 °C to 50 °C in PCR machine to form a double strand and ligated to pAcGFP1-N3 vector, which was precut by HindIII and BamHI. As shown in Fig. 1B, the 506-MMP1 (sequence 506–530) had no translation initiation codon “ATG” and its last 2 codes were “AT”. One cytidylic acid “C” was extended at the 3′-end of 506-MMP1F′ oligonucleotide, as indicated by “q”, to avoid translation initiation codon “ATG” been created after ligated with BamHI, since the created “ATG” would be used as translation initiation codon, and frame shift mutation would happen in the following codons of AcGFP1. As shown in Fig.