Platinum sybr green qpcr supermix for icycler

qRT-PCR was carried out in a iCycler IQ apparatus (Bio-Rad) using Platinum SYBR Green qPCR SuperMix for iCycler (Life Technologies) in accordance with the manufacturer's recommended protocol. Reactions were carried out using 2 μl of cDNA, in a total reaction volume of 26 μl. The genes measured for L. asiaticus included prbP (CLIBASIA_01510), L25 (CLIBASIA_01515), rplK (CLIBASIA_00130), rpsJ (CLIBASIA_00735), gyrA (CLIBASIA_00325), and 16S rRNA (CLIBASIA_r05785). The mRNA levels of rplK, CLIBASIA_r05785, rpsJ and prbP genes were normalized to the abundance of the plant genes cox2 and 18S rRNA. The expression of each plant control gene was previously examined in absence and presence of tolfenamic acid (data not shown). Quantitative reverse transcription-PCR primers are described in detail in Table 2.

L. crescens cells were cultured in broth with hexestrol (25 µM), phloretin (50 µM), or benzbromarone (50 µM) when required. The cells were collected by centrifugation at 4°C when OD₆₀₀ = 0.3 (mid-exponential phase). Total RNA was subsequently isolated with RiboPure-Bacteria (Ambion) in accordance with the manufacturer's protocol. cDNAs were synthesized with the Superscript first-strand synthesis kit (Life Technologies) in accordance with the manufacturer's instructions and stored at −80°C prior to use. Real-time quantitative PCR (qRT-PCR) was carried out in a iCycler IQ apparatus (Bio-Rad) using Platinum SYBR Green qPCR SuperMix for iCycler (Life Technologies) in accordance with the manufacturer's recommended protocol. Primers used for the qRT-PCR are described in further detail on Table 1. The RNA polymerase sigma factor rpoD, 50S ribosomal protein L10, 50S ribosomal protein L12 genes, (B488_13350, B488_08460, B488_08450, respectively), and 16S ribosomal RNA were used as internal controls.