Quantifying Staphylococcus aureus RNA Levels

The eluted bacterial total RNA was qualified with a 2100 Bioanalyzer (Agilent) using an RNA 6000 Pico chip and analyzed by quantitative reverse transcription PCR (RT-qPCR) using the CFX Connect Real-Time PCR system (Bio-Rad) as follows. To convert RNA to cDNA and then quantify the cDNA in a single step, a Luna® Universal One-Step RT-qPCR Kit (New England BioLabs) was used with a FAM dye-labeled probe for ribonuclease P RNA (rnpB) in S. aureus (Taqman® gene expression assays, ID number: Ba04646259_s1, Thermo Fisher Scientific) following the manufacturer's protocol. RT-qPCR was performed with 1 cycle of 95 °C for 10 min and 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. Quantification of gene expression was based on the cycle quantification (Cq) value compared with that of the positive controls extracted by the RNeasy Mini Kit from Qiagen. The delta Cq was calculated as (Cq of captured sample on chip) - (Cq of control sample) using CFX Maestro 1.1 software (Bio-Rad) and Microsoft Excel (Microsoft Corp., USA). The relative quantity of rnpB gene expression was determined by a delta-delta Cq calculation as 2^(-(treated sample delta Ct-control sample delta Ct)) 29 (link).