Fig 5.

Dose response allele ratios and relative mRNA levels of COL1A1 and COL1A2 indels at 72 hours. (A) Ratio of insertion(i)/non-insertion(ni) alleles for cell populations treated with either of the siRNAs targeting COL1A1 indel rs3840870 or COL1A2 indel rs3917 72 hours post transfection. A range of siRNA concentrations was used: 0.025µg, 0.05µg 0.1µg, 0.2µg, 0.4µg and 0.6µg for indel-targeting siRNAs and for negative controls (NC) 0.6µg was used. The software PeakPicker was used to calculate the heights of the same three peak-pairs in all cDNA chromatograms and values were normalized to negative control treated cells, with the mean defined as 1. Error bars indicate standard deviation. The allele ratios for non-insertion treated siRNAs have been inverted to enable presentation of results in the same chart. Color of bars denotes category of siRNA (NC-red, i-green and ni-blue). A two-tailed T-test comparing negative controls with each type and dose of siRNA was performed. * = p < 0.05; ** = p < 0.001 and n.s. = not significant. (B) Average COL1A1 and COL1A2 mRNA levels calculated from the two probes used per collagen gene in relation to levels of control genes (GAPDH and ACTB) measured by quantitative PCR in negative controls (NC) and siRNA treated wells. The mean ratio of COL1A and COL1A2 to either reference gene in negative control wells was defined as 1. Error bars indicate standard deviation. Pattern of bars denote reference gene (solid for GAPDH and striped for ACTB) used in quantitative PCR and color denotes the category of used siRNA (NC-red, i-green and ni-blue).