For each round of SCOTS, 3 μg cDNA
samples were denatured at 98 °C for 3 min and normalized by self-hybridization, and hybridized subsequently PLX4032 clinical trial at 65 °C for 24 h with 0.6 μg photobiotinylated C51-17 genomic DNA that had been blocked previously with 5 μg 16S and 23S rRNA genes. The cDNAs were captured by streptavidin-coupled magnetic beads (Dynal M280, Invitrogen) according the manufacturer’s instructions. After elution, the cDNAs were re-amplified by PCR using the primer, SCOTS-01 or SCOTS-02. For each growth condition, in the first round of normalization, 10 separate samples of the cDNA mixture were captured by hybridization in parallel reactions and the 10 amplified cDNA preparations were combined for further procedures. To identify cDNA molecules that represented transcripts from genes that were specific to or upregulated in expression during growth of P. multocida in the liver, an enrichment process was included in the experiments as described previously (Hou et al., 2002). The final captured cDNAs were cloned into the pMD18-T vector (TaKaRa), and
the white clones on the X-gal plates were subjected to a Southern dot blot and sequenced using the standard Z-VAD-FMK nmr Sanger method. Database searches and DNA and protein similarity comparisons were carried out using the blast algorithm from the National Center for Biotechnology Information at the National Library of Medicine (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi). Five microliters of PCR product from each clone was denatured by boiling and transferred onto a positively charged nylon membrane (Millipore, Billerica, Morocco). The cDNA mixture was amplified from three rounds of normalization using the specific primer SCOTS-01 or SCOTS-02 and labeled with digoxigenin (DIG)-labeling mix to form probes. Membranes were fixed, prehybridized and hybridized at 42 °C, and hybridization signals were detected using the DIG Detection Kit (Roche, Germany) according to
the manufacturer’s instructions. Total RNAs isolated from bacterial pellets and infected Paclitaxel cost livers were reverse transcribed as the same primers used earlier. The real-time PCR assay was performed using SYBR-Green dye (TaKaRa). Specific gene primers were designed for the qRT-PCR, and the sequences are shown in Table 1. Each 20 μL reaction included 100 ng cDNA, 200 nmol of each primer and 10 μL 2× SYBR-Green dye. The following cycles were performed: 95 °C for 3 min for the hot-start, followed by 40 cycles of 95 °C for 15 s, 65 °C for 30 s, and 72 °C for 45 s. The Ct value for the 40 cycles was recorded, and qRT-PCR analysis of P. multocida RNA derived from in vivo and in vitro cultures was performed for the test genes and the internal control of the 16S rRNA gene in triplicate. The relative level of expression was calculated using the method.