Mx3000p real time pcr system cycler

For RNA extraction, bacteria were cultured in rich media up to an exponential growth phase. Total RNA was extracted using Epicentre ^® Master Pure™ Kit (Illumina). One microgram of RNA was treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) and subjected to retro-transcription with Super Script III Reverse Transcriptase and random hexadeoxynucleotides (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. cDNAs were then amplified with FastStart Universal SYBR Green Master (Roche, Basel, Switzerland) using the Mx3000P Real Time PCR System cycler (Agilent Technologies, Santa Clara, CA, USA). The results of each target were normalized to B. suis Translation Initiation Factor-1 (if-1, BR0249) mRNA (Eskra et al., 2001 (link)). The relative expression levels of the target genes were calculated according to the comparative critical threshold (ΔΔC_T) method (Livak and Schmittgen, 2001 (link)). Three biological samples were measured for each strain. Oligonucleotides used are listed in Table S1.

Total wild-type or ΔmliR B. argentinensis JUB59 RNA from three independent cultures was isolated as described above. Subsequently, reverse transcription was performed using the first−strand SuperScript III cDNA kit (Invitrogen) and random decamer experiments primers (Invitrogen) in the presence of RNasin ribonuclease inhibitor (Promega). Transcript levels were measured in a Mx3000P Real Time PCR System cycler (Agilent Technologies, Santa Clara, CA, United States) using cDNAs as template, FastStart Universal SYBR Green Master (ROX) (Roche), and the RT-qPCR primers listed in Supplementary Table S1. Transcript levels were normalized to that of the serine hydroxymethyltransferase A (glyA) housekeeping as previously reported for members of the Flavobacteriaceae family (Thomas et al., 2011a (link)).