After 1 h of polymerization, PCR samples were loaded into the wells, and electrophoresis was performed for 16 h at 70 V PHA-848125 nmr in 1 × TAE buffer at a constant temperature of

60°C by using the Dcode system (Biorad). DGGE gels were stained for 30 min with 1 × SYBR® Gold (Molecular Probes) in 1 × TAE buffer. This was followed by visualization of DGGE band profiles under UV light. Digital capturing was performed by using a Geldoc XR camera system (Biorad) combined with the Quantity One software package (Biorad). By including a standard reference every six lanes in each DGGE gel, it was possible to digitally normalize the gel profiles by comparison with a standard pattern using the BioNumerics software, version 2.5 (Applied Maths, St.-Martens-Latem, Belgium). This normalization enabled comparison between DGGE profiles from different gels provided that these were run under comparable denaturing and electrophoresis conditions. The digitalized data were exported as an excel file for further statistical analysis according to Gafan et al. [13]. In this file, each sample represented buy Bortezomib 1 row and each band was assigned to a unique band position (column) with 1 indicating presence and 0 indicating absence of a band at that position. API was used as outcome parameter. The data were first analysed using

Chi-square tests with a cut-off for significance at 5% to reduce the number of bands included in multivariate analysis. Significant bands (absent coded as 0, present coded as 1) were included as explanatory variables in a multivariate CA-4948 price logistic regression

analysis (API as Carnitine palmitoyltransferase II dependent variable, negative coded as 0, positive as 1) for each primer set separately. The estimated odds ratio was calculated for each band in the logistic regression model. Bands remaining significantly associated with the API index in this model were adjusted for 5 known asthma confounders (exclusive breast feeding, maternal smoking during pregnancy, infant use of antibiotics at age of 3 weeks, parental socio-economic status and gender) in a second logistic regression model. The statistical analysis was conducted using SPSS version 15 (Chicago, USA). Significant bands in this second model were identified after excision of the band from the gel and overnight incubation in TE buffer at 4°C. After extraction of the band it was reamplified with the corresponding primer set and reanalysed in DGGE together with the original fecal sample to confirm if the correct band was extracted. This process was repeated 2-3 times until a single band was obtained. This band was subsequently sequenced without additional cloning by the Genetic Service Facility (VIB, University of Antwerp) with a capillary sequencer (Applied Biosystems 3730 DNA analyser) using the corresponding forward and reverse primers without the GC clamp. In all cases this procedure resulted in a pure sequence product from the excised band.