Journal: Biotechnology Reports
Article Title: Effect of small interfering RNAs on matrix metalloproteinase 1 expression
Figure Lengend Snippet: Analysis of endogenous MMP1 gene transcription by quantitative-PCR. MeWo cells were treated with different concentration (0, 10, 30, 50, 70, 90 nM) of target designed siRNA (506 siRNA and 859 siRNA) and incubated for 24 h. After incubation, the total RNA was extracted using TRIzol Reagent. The cDNA was synthesized using 1 μg of total RNA by reverse-transcription using iScript™ cDNA synthesis kit. MMP1 and GAPDH gene expression was carried out using Quantitative-PCR/iQ™ SYBR ® Green Supermix (Bio-rad, California). (A) was the 1.5% agarose gel electrophoresis assay of the PCR products of different siRNA treatments. (B) was the quantified results of the expression quantity of relative expression of the MMP1 mRNA normalized against GAPDH mRNA and compared to blank (without treatment of siRNA).
Article Snippet: For real-time PCR analysis of MMP1 and GAPDH gene expression was carried out using iQ™ SYBR® Green Supermix (Bio-rad, California), the primers used were: • MMP1 forward: AGTCAAGTTTGTGGCTTATGGA • MMP1 reverse: TTGTCACTGAAGCTGCTCTC • GAPDH forward: GTCGGAGTCAACGGATTT • GAPDH reverse: CAACAATATCCACTTTACCAGAG 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.
Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Incubation, Synthesized, Expressing, SYBR Green Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction