Toward the pre-16S leader region known as the external transcribed spacer (ETS1) (Fig. 1A). The mixture (RNA and primers) was denatured at 65uC for 5 minutes, rapidly cooled on wet ice for 2? minutes, and supplemented with 4 mL 5X First Strand buffer, 2 mL 0.1 M DTT, 1 mL 10 mM dNTPs, 2 1081537 mL DEPC-treated H2O, and 1 mL SuperScript III RT enzyme (Invitrogen Cat# 18080-044). These reactions were incubated at 50uC for 50 minutes. The [DTrp6]-LH-RH supplier remaining RNA was hydrolyzed with NaOH, and the more stable cDNA cleaned using Qiagen’s PCR purification kit and eluted in 50 mL elution buffer. Quantitative PCR (qPCR) amplification of cDNA or genomic DNA (gDNA) was run on an ABI Prism FAST RT-7500 PCR system using the Applied Biosystems Power SYBR Green mix (4367659). Samples were run in triplicate along a range of appropriate dilutions; briefly, 2 mL templates were added to 0.25 mL (7.5 nmoles) of each primer, 10 mL 26SYBR mix, and 7.5 mL H2O to a final volume of 20 mL per well. The primer sets bridged the earlier-described junction between ETS1 and the mature 16S rRNA (Figure 1). Standard curves were generated using 10-fold serially-diluted genomic DNA (gDNA) for quantification of pre-rRNA and gDNA amplified by using the same primers. The gDNA used for standard curves was extracted from bacterial cultures by using commercial DNA purification kits. Its quantity (in ug/mL) and purity was assessed spectrophotometrically. Reaction conditions were as follows: 10 minutes 95uC, 40 cycles of (15 s at 95uC, 30 s at 57uC, 30 s at 72uC) using `9600 emulation.’ ABI’s software (7500 Fast System SDS v1.4.025, and v2.0.5) was used to automatically set Ct threshold values, except when manual adjustment into the exponential range wasViability Testing by Pre-rRNA AnalysisFigure 1. Reverse transcription and PCR primers. A: Oligonucleotides used for priming reverse transcription of pre-rRNA (cDNA synthesis), and forward and reverse PCR primers for amplifying cDNA. B: Specificity of PCR primer sets designed for S. aureus, A. baumannii, P. aeruginosa, and MTBC. Each primer set was used in endpoint PCR conducted on purified DNA from all the four organisms. Key: L, 1 Kb+ DNA ladder; 1, S. aureus DNA; 2, A. baumannii DNA; 3, P. aeruginosa DNA; 4, M. tuberculosis DNA; 5, negative control (water). Lowest three bands of ladder are 100, 200, and 300 bp. A repeat experiment yielded identical results. doi:10.1371/journal.pone.0054886.gnecessary. The results were exported to a spreadsheet for analysis. From separate gDNA standard curves in each experiment, qPCR efficiencies were calculated [10(21/slope) 21]. To calculate ratios of pre-rRNA to gDNA (P:G) in nutritionally stimulated and non-stimulated samples, inputted pre-rRNA and gDNA were quantified relative to a gDNA standard curve amplified with the same primers. Standard deviations were calculated from all possible ratiometric permutations.Serum Acclimation Time CourseIn a second set of experiments, human serum was inoculated with a smaller number of cells (,1E5/mL) and acclimated in serum for shorter periods (as little as four hours). A. baumannii, P. aeruginosa, and S. aureus were cultured overnight as above. Cells were Triptorelin site removed, washed and resuspended in PBS for OD600 measurement. Triplicate 5 mL pre-warmed serum suspensions of each organism were prepared at ,16105 CFU/mL in 14 mL round-bottom tubes and incubated shaking at 37uC. After acclimation in serum for 4, 24, and 168 hours, sample collection and nutritional stimulation were i.Toward the pre-16S leader region known as the external transcribed spacer (ETS1) (Fig. 1A). The mixture (RNA and primers) was denatured at 65uC for 5 minutes, rapidly cooled on wet ice for 2? minutes, and supplemented with 4 mL 5X First Strand buffer, 2 mL 0.1 M DTT, 1 mL 10 mM dNTPs, 2 1081537 mL DEPC-treated H2O, and 1 mL SuperScript III RT enzyme (Invitrogen Cat# 18080-044). These reactions were incubated at 50uC for 50 minutes. The remaining RNA was hydrolyzed with NaOH, and the more stable cDNA cleaned using Qiagen’s PCR purification kit and eluted in 50 mL elution buffer. Quantitative PCR (qPCR) amplification of cDNA or genomic DNA (gDNA) was run on an ABI Prism FAST RT-7500 PCR system using the Applied Biosystems Power SYBR Green mix (4367659). Samples were run in triplicate along a range of appropriate dilutions; briefly, 2 mL templates were added to 0.25 mL (7.5 nmoles) of each primer, 10 mL 26SYBR mix, and 7.5 mL H2O to a final volume of 20 mL per well. The primer sets bridged the earlier-described junction between ETS1 and the mature 16S rRNA (Figure 1). Standard curves were generated using 10-fold serially-diluted genomic DNA (gDNA) for quantification of pre-rRNA and gDNA amplified by using the same primers. The gDNA used for standard curves was extracted from bacterial cultures by using commercial DNA purification kits. Its quantity (in ug/mL) and purity was assessed spectrophotometrically. Reaction conditions were as follows: 10 minutes 95uC, 40 cycles of (15 s at 95uC, 30 s at 57uC, 30 s at 72uC) using `9600 emulation.’ ABI’s software (7500 Fast System SDS v1.4.025, and v2.0.5) was used to automatically set Ct threshold values, except when manual adjustment into the exponential range wasViability Testing by Pre-rRNA AnalysisFigure 1. Reverse transcription and PCR primers. A: Oligonucleotides used for priming reverse transcription of pre-rRNA (cDNA synthesis), and forward and reverse PCR primers for amplifying cDNA. B: Specificity of PCR primer sets designed for S. aureus, A. baumannii, P. aeruginosa, and MTBC. Each primer set was used in endpoint PCR conducted on purified DNA from all the four organisms. Key: L, 1 Kb+ DNA ladder; 1, S. aureus DNA; 2, A. baumannii DNA; 3, P. aeruginosa DNA; 4, M. tuberculosis DNA; 5, negative control (water). Lowest three bands of ladder are 100, 200, and 300 bp. A repeat experiment yielded identical results. doi:10.1371/journal.pone.0054886.gnecessary. The results were exported to a spreadsheet for analysis. From separate gDNA standard curves in each experiment, qPCR efficiencies were calculated [10(21/slope) 21]. To calculate ratios of pre-rRNA to gDNA (P:G) in nutritionally stimulated and non-stimulated samples, inputted pre-rRNA and gDNA were quantified relative to a gDNA standard curve amplified with the same primers. Standard deviations were calculated from all possible ratiometric permutations.Serum Acclimation Time CourseIn a second set of experiments, human serum was inoculated with a smaller number of cells (,1E5/mL) and acclimated in serum for shorter periods (as little as four hours). A. baumannii, P. aeruginosa, and S. aureus were cultured overnight as above. Cells were removed, washed and resuspended in PBS for OD600 measurement. Triplicate 5 mL pre-warmed serum suspensions of each organism were prepared at ,16105 CFU/mL in 14 mL round-bottom tubes and incubated shaking at 37uC. After acclimation in serum for 4, 24, and 168 hours, sample collection and nutritional stimulation were i.
