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Role of Intestinal Microbiota in Ulcerative Colitis

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Appendix 2<br />

Methodology part<br />

Quantification <strong>of</strong> eight bacterial taxa to validate the comb<strong>in</strong>ed DNA extraction method<br />

Appendix 2<br />

Quantitative Real‐Time PCR was performed us<strong>in</strong>g primers target<strong>in</strong>g Bifidobacterium spp. (Delroisse<br />

et al., 2008), Lactobacillus spp. (Walter et al., 2000;Heilig et al., 2002), Coccoides leptum subgroup<br />

(Walter et al., 2000;Matsuki et al., 2004;Shen et al., 2006), Firmicutes (Guo et al., 2008),<br />

Desulfovibrio spp. (Fite et al., 2004), Bacteroidetes (Guo et al., 2008), Bacteroides spp. (Ahmed et<br />

al., 2007), Alistipes spp. (Paper 1) and. DNA extraction and qPCR procedure are performed as<br />

described <strong>in</strong> Paper 2‐6.<br />

Log(Relative quantity <strong>of</strong> 16S rRNA)<br />

5<br />

4<br />

3<br />

2<br />

1<br />

0<br />

Bifidobacterium spp.<br />

Lactobacillus spp.<br />

Bifidobacterium spp.<br />

Lactobacillus spp.<br />

C. leptum subgroup<br />

Gram-positive bacteria<br />

C. leptum subgroup<br />

Firmicutes<br />

Firmicutes<br />

Log(Relative quantity <strong>of</strong> 16S rRNA)<br />

5<br />

4<br />

3<br />

2<br />

1<br />

0<br />

Desulfovibrio spp.<br />

Desulfovibrio spp.<br />

Bacteroidetes<br />

Bacteroidetes<br />

Gram-negative bacteria<br />

Bacteroides spp.<br />

Bacteroides spp.<br />

Alistipes spp.<br />

Alistipes spp.<br />

Relative quantities <strong>of</strong> gene targets encod<strong>in</strong>g 16S rRNA from four Gram‐positive bacteria and four<br />

Gram‐negative bacteria <strong>in</strong> either 0 hours fermentation samples (white) or fecal samples (hatched).<br />

Samples were obta<strong>in</strong>ed from three healthy subjects. Error bars represent averages ± standard<br />

deviation <strong>of</strong> the three different subjects.<br />

The data was log‐transformed before statistical analysis to obta<strong>in</strong> normal distribution. Two‐tailed<br />

pair wise t‐test was used to analyze difference <strong>in</strong> samples. Tests were considered statistically<br />

significant if P‐values lower than 0.05 were obta<strong>in</strong>ed.<br />

153

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