geNorm analysis of colonic HT-29 (A and C) and vaginal VK2/E6E7 (B and D) cell lines.

<p>The geNorm stability (M) values are shown in order of increasing stability for both colonic HT-29 (A) and vaginal VK2/E6E7 cell lines (B). The steeper the line between two adjacent genes, the greater the impact removal of this gene has on overall reference gene stability. The geNorm variation (V) assessment is also summarised for colonic HT-29 (C) and vaginal VK2/E6E7 (D) cell lines. A lower value in this analysis indicates less variation and increased accuracy of normalisation. The cut off at which addition of further reference genes is not presumed to improve normalisation ability is 0.15, and is shown by the dashed red line.</p

Range of C_q values for the colonic HT-29 (A) and vaginal VK2/E6E7 (B) cell lines.

<p>The range of C_q values as measured by qPCR for both the HT29 (A) and VK2/E6E7 (B) cells. The plots highlight the median (centre line), maximum and minimum (whiskers), and 1^st and 3^rd quartile marks (boxes) of the data. All C_q values measures were within the bounds of the linear dynamic range as determined by standard curve analysis.</p

Simulation of hypGOI expression study in VK2/E6E7 cells treated with <i>Lactobacillus acidophilus</i> NCFM.

<p>The columns show the effect of treatment on a hypothetical gene of interest (hypGOI) when normalising against the different reference genes based on expression levels determined in this study. The most stable gene assessed (<i>RPLP0</i>) was used as the standard for the target fold changes, which were set at two-fold decrease (A), two-fold increase (B), five-fold decrease (C), and five-fold increase (D). The three lower-ranked reference genes assessed (<i>PGK1</i>, <i>DICER1</i>, and <i>MVK</i>) are shown in order of decreasing stability as determined in this study. The control expression level was set at one as an arbitrary reference point, and is shown by the dashed red line. Error bars indicate the standard deviation.</p

Mean standard deviation (s.d.) of reference genes using the comparative ΔC_q method.

<p>Mean standard deviation (s.d.) of reference genes using the comparative ΔC_q method.</p

Primer efficiencies and linear dynamic range.

<p>Primer efficiencies and linear dynamic range.</p

Simulation of hypGOI expression study in HT-29 cells treated with <i>Lactobacillus acidophilus</i> NCFM.

<p>The columns show the effect of treatment on a hypothetical gene of interest when normalising against the different reference genes based on expression levels determined in this study. The most stable gene assessed (<i>PGK1</i>) was used as the standard for the target fold changes, which were set at two-fold decrease (A), two-fold increase (B), five-fold decrease (C), and five-fold increase (D). The three lower-ranked reference genes assessed (<i>MVK</i>, <i>ACTB</i>, and <i>DEFB1</i>) are shown in order of decreasing stability as determined in this study. The control expression level was set at one as an arbitrary reference point, and is shown by the dashed red line. Error bars indicate the standard deviation.</p

Comparison of reference gene rankings based on cell type and bacterial challenge.

<p>Gene names are around the outside of the wheel and follow the radial lines. The numbered concentric lines indicate the geometric ranking. The further away from the centre the line is, the more stable the reference gene. Each data set is plotted with a different colour. The degree to which the coloured lines overlap indicate how well the data sets correlate to each other. <b>A.</b> Colonic HT-29 cells. Blue: combined data set. Red: <i>Lactobacillus acidophilus</i> NCFM used as the commensal strain. Green: <i>Lactobacillus rhamnosus</i> GR-1 used as the commensal strain. <b>B.</b> Vaginal VK2/E6E7 cells. Blue: combined data set. Red: <i>L. acidophilus</i> NCFM used as the commensal strain. Green: <i>L. rhamnosus</i> GR-1 used as the commensal strain. <b>C.</b> Comparison of combined data sets of the HT-29 and VK2/E6E7 cell lines. Blue: HT-29 cells. Red: VK2/E6E7 cells.</p

Summary of key statistics after BestKeeper analysis.

<p>The standard deviation (s.d.) corresponds to the individual gene stability and the coefficient of correlation, with its respective p-value, corresponds to how closely the candidate reference gene resembles the BestKeeper ideal normalisation factor after repeated pair-wise analysis. The power value is determined by regression analysis, using fold change (x-fold) as a reference point, and a smaller value indicates a better reference candidate. Values for genes eliminated in earlier stages of analysis are not shown.</p><p>Summary of key statistics after BestKeeper analysis.</p

Comprehensive ranking of reference gene candidates by calculation of a geometric mean.

<p>Comprehensive ranking of reference gene candidates by calculation of a geometric mean.</p