Sybr green pcr reagent

20 healthy adults were recruited for the study. Exclusion criteria were use of any antimicrobials within the last two weeks and use of cosmetics within the preceding 24 h. The participants were unrelated. Separate mucosal swabs were collected from the face and armpit of each participant, and the swabs were immediately submerged in 1 ml normal saline. All samples were kept at −80°C until further use. For RNA isolation, swab samples were vortexed for 2 min and centrifuged at 13,000 × g for 10 min at 4 °C. The bacterial pellets were lyzed with a Mini-Beadbeater (Biospec Products) at maximum speed for 30 s. After incubation on ice for 5 min, the samples were centrifuged; and the supernatants were used to isolate total RNA using a Qiagen RNeasy kit (Qiagen 74106) according to the manufacturer’s instructions. Genomic DNA was removed by a wipeout buffer (Qiagen 205311) and approximately 0.2 μg of total RNA was converted to cDNA using a reverse transcription kit (Qiagen 205311). The obtained cDNA was used as a template for qRT-PCR using a SYBR-green PCR reagent (Roche). See Table S2 for oligonucleotides. The 16S rRNA gene of S. epidermidis was used as a housekeeping gene control. Reactions were performed in MicroAmp Optical 96-well reaction plates and fluorescence signals were detected by a 7500 Sequence Detector (Applied Biosystems).

For measurement of gene expression of inflammatory cytokines, monolayers of A549 cells grown in DMEM with 10% FBS were exposed to 1 × 10⁴ CFU of S. salivarius or S. epidermidis isolates for 24 h with or without addition of A. alternata at a final concentration of 35 ng/ml, and cells were washed and harvested. Alternatively, DMEM with 10% FBS was inoculated with 1 × 10⁴ CFU of S. salivarius or S. epidermidis and incubated for 24 h, then culture filtrates were collected and added to the monolayer of A549 cells for another 24 h. Real-time quantitative PCR (qRT-PCR) was used to measure gene expression. To that end, total RNA was isolated from cells using a QIAGEN RNeasy kit (QIAGEN 74106) according to the manufacturer’s instructions. After treatment with genomic DNA wipeout buffer (QIAGEN 205311), approximately 1 μg of total RNA was reverse-transcribed with a Reverse Transcription Kit (QIAGEN 205311), and the obtained cDNA was used as a template for qRT-PCR using SYBR-green PCR reagent (Roche). The primers used are listed in Supporting Information Table 1. The gapdh gene was used as a housekeeping gene control. Reactions were performed in MicroAmp Optical 96-well reaction plates using a 7500 Sequence Detector (Applied Biosystems).