, 1998), calls for methods
with both high sensitivity toward low-abundant taxa and a high dynamic range. Real-time qPCR analysis has these properties and is a relatively simple and affordable technique, which offers a high degree of reproducibility and specificity and has thus found extensive use in the quantitative analysis of gut bacterial populations (Huijsdens et al., 2002; Bartosch et al., 2004; Gueimonde et al., 2004; Matsuki et al., 2004; Haarman & Knol, 2006; Larsen et al., 2010; Petersen et al., 2010; Combes et al., 2011; Vigsnæs et al., 2011). Analysis of multiple bacterial targets using qPCR is, however, to some extent technically limited by the fact that each primer set is associated with specific temperature cycling conditions, necessitating separate DAPT qPCR setup for each target. Furthermore, because of varying PCR efficiency, amplicon lengths, and GC-content (Colborn et al., 2008), INK 128 ic50 the generation of standard curves for each target gene is normally required in order to provide absolute quantification. In many metagenomic studies however, such absolute quantifications
may not be essential, as focus may primarily be placed on identifying specific changes occurring in microbial communities of the individuals undergoing a certain intervention (e.g. the individuals in a group given probiotics as compared to a control group), rather than obtaining information on absolute quantities or abundances of different bacterial taxonomical groups (Larsen et al., 2011). In the present study, we develop and validate a real-time qPCR platform, GUt Low-Density Array (GULDA), that allows the simultaneous
relative quantification of 31 relevant microbial 16S rRNA gene targets in two extracted community DNA samples, with four technical replicates all performed on one 384-well plate using a universal thermocycling program. Relative quantities of specific rRNA genes are calculated using a universal bacterial 16S rRNA gene as reference, and fold-changes for each microbial group are determined without the use of standard curves. Genomic DNA from a total of 27 bacterial strains and one archaeal strain was either obtained directly from Deutsche Sammlung von Mikroorganismen und Zellkulturen gmbH, Germany (DSM), as extracted DNA or extracted Rebamipide from pure cultures originating from either DSM or The American Type Culture Collection, USA (ATCC; Fig. 1). Fecal samples obtained from six randomly selected infants from the SKOT cohort (Madsen et al., 2010) at both 9 and 18 months of age were selected, and community DNA was extracted on the Maxwell® 16 system using the Maxwell® 16 DNA Tissue DNA purification kit (Promega Biotech AB, Sweden). In all cases, the DNA concentrations were determined fluorometrically (Qubit® dsDNA HS assay; Invitrogen) and adjusted to 1 ng μL−1 prior to use as template in qPCR.