Bio plex 200 system array reader

Total RNA was isolated using RNA-Bee reagent (Tel-Test Inc., Friendswood, TX) and concentrations were quantified spectrophotometrically at 260 nm. Integrity and quality of RNA samples were evaluated by formaldehyde-agarose gel electrophoresis. Mutiplex suspension arrays (Panomics/Affymetrix, Fremont, CA) were performed to quantify the mRNA expression. Individual gene accession numbers can be accessed at www.panomics.com (sets #21330 and #21383). Samples were analyzed using a Bio-Plex 200 System Array reader with Luminex 100 xMAP technology, and the data were acquired using Bio-Plex Data Manager version 5.0 (Bio-Rad, Hercules, CA). Assays were performed according to the manufacturer’s protocol. The mRNA data were normalized to Rpl13a mRNA and presented as fold change relative to the WT control group.

Messenger RNAs for mouse genes involved in BA synthesis and hepatic BA transporters were quantified using the QuantiGene Plex 2.0 assay (Panomics/Affymetrix, Fremont, CA). Individual bead-based oligonucleotide probe sets were designed by Panomics/Affymetrix, Inc. using previously published NCBI gene accession numbers (panel IDs: 21140, 21065, and 21150). Assays were performed following the manufacturer’s protocol (Panomics/Affymetrix, Fremont, CA). Briefly, isolated RNA (250 ng) was hybridized overnight at 54 °C to specific oligonucleotide sequences, corresponding to the genes of interest that were bound to capture beads. A linearity test was done prior to the analysis. The following day the hybridized RNA, oligos, and capture beads were pipetted onto a filter plate. The complex was then sequentially hybridized to the pre-amplifier, followed by the amplifier, and lastly the biotinylated label probe (each for 1 h at 50 °C on a shaking platform). Streptavidin–phycoerythrin was then allowed to bind for 30 min at room temperature on a shaking platform. Fluorescence was detected and analyzed with a Bioplex 200 system array reader with Luminex xMAP technology (Bio-Rad, Hercules, CA). The housekeeper gene, Gapdh, was used to normalize the data. The data are expressed as a ratio of the relative light units of the target gene mRNA relative to Gapdh mRNA.