Role of Intestinal Microbiota in Ulcerative Colitis

Methodology part
6. Methodology, considerations and choices
extraction methods and two of the major concerns important for accuracy of ecological studies
are cell lysis efficiency and quality of DNA extracts (Li et al., 2003). DNA from organisms, which cell
walls are easily lysed (Gram‐negative bacteria) can be sheared, if the extraction conditions are too
rough. However, DNA can be difficult to recover from organisms with rigid cell walls (Gram‐
positive bacteria), if the cell lysis is insufficient. Additionally, it is important that extracted DNA is
without unfavorable substances, because they may inhibit e.g. PCR amplification (Monteiro et al.,
1997;Lantz et al., 1997). Mechanical disruption of human fecal samples prior extraction using
QIAamp DNA stool MiniKit has previously shown to improve the amount and quality of DNA (Li et
al., 2007;Smith et al., 2011). Thus, combination of these two methods allows effective lyses of
bacteria cells and removal of organic contaminants leading to high quality DNA that can be further
analyzed using down‐stream molecular techniques (Li et al., 2003). Prior to the in vitro studies
performed in the experimental part of this thesis (Paper 2‐6), the combined extraction method
was validated by extracting DNA from both fermentation samples at 0 hours and fecal samples
(n=6, healthy subjects) to ensure that this method could yield the same amount of DNA from the
different sample types. Eight bacterial groups (four Gram‐negative and four Gram‐positive
bacteria) were quantified by qPCR to compare DNA extracted from either fermentation or fecal
samples. The results demonstrated no significant difference between the two sample types for the
eight bacterial taxa (Appendix 2). Hence, the combined extraction method was used to extract
DNA from both fecal and fermentation samples in the experiments of this thesis (Paper 2‐6).
6.5. Quantitative Real-Time PCR
QPCR is a useful tool for quantitative detection of target sequences in e.g. bacterial community,
due to its high sensitivity. In qPCR, the accumulation of product amplification is detected by the
presence of a fluorescence reporter. The quantification is done by measuring the number of cycles
required for fluorescent signal to reach a threshold level. The cycle number is proportional to the
number of copies of DNA templates in a sample (Saunders, 2009). Different fluorescence reporters
can be used in qPCR assay. One approached is the specific hybridization of a duallabelled TaqMan
probe to PCR product. Another is based upon the binding of the fluorescent dye SYBR‐Green into
the PCR product (Ponchel et al., 2003). SYBR‐Green was used as fluorescent reporter in the qPCR
assays in Paper 1‐6.
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