Computational protocol: A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes

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Protocol publication

[…] Complementary RNA (cRNA) was produced from the RNA samples and fluorescently labeled using the MessageAmp™ II-Bacteria kit (Ambion) according to the manufacturer's instructions with either Cy3-CTP or Cy5-CTP (Perkin Elmer) as the fluorescent label. Specific activity and cRNA concentrations were determined using a NanoDrop ND-1000 spectrophotometer. The labeled samples were stored at −70°C until use. Dual-color DNA microarray hybridizations were performed using a custom Agilent 8x15K slide containing probes for the 4196 chromosomal and plasmidial Y. pestis CO92 genes and pseudogenes [Accession no. NC_003143.1 NCBI; (Parkhill et al., )]. Hybridization and scanning were performed as suggested by Agilent. The slides were scanned in the Agilent DNA microarray scanner G2505B. Images were analyzed, and data were extracted using the Agilent Feature Extraction (FE) software (version 9.5.1.1), with linear and lowess normalization. Statistical analysis was performed using the Limma (Linear Models for Microarray Data) package from the Bioconductor project (http://www.bioconductor.org). The processed signal from the FE was read into Limma using the “read.maimages” function. Background subtraction and lowess normalization were performed for each array. Quantile normalization was applied between arrays. The Benjamini-Hochberg false discovery rate (FDR) was used to correct for multiple comparisons. The fold change (FC) in each gene was calculated as the median FC value measured for 3–5 different or replicated probes representing the same gene. Each experiment comprised 4 samples representing 4 different ciprofloxacin concentrations and labeled with Cy3-CTP that were co-hybridized with the growth control sample, which was not exposed to ciprofloxacin and was labeled with Cy5-CTP. Each slide was hybridized with samples originating from two independent biological experiments representing the same time point, performed with Cy3-Cy5 dye swap. The results are available online (http://www.ncbi.nlm.nih.gov/geo/), GEO ID: GSM2101145. [...] One-step qRT-PCR was performed in a 50-μl reaction mixture containing 1 ng of total RNA as the template, 1 × buffer (0.5 M KCl, 0.1 M Tris-HCl, pH 8.8 [Bio-Rad], 1 μM SuperROX® [Biosearch Technologies]), 0.6 μM each primer, 0.3 μM TaqMan probe, 0.2 mM dNTP, 3 mM MgCl2, 0.5 μl of Sensiscript reverse transcriptase (QIAGEN), 2 U of Taq DNA polymerase (Promega) and 0.4 μl of JumpStart Taq antibodies (Sigma). Gene-specific primers and TaqMan probes (5′ 6-FAM; 3′ BHQ-1) were designed based on the Y. pestis CO92 genomic sequence (NC_003143.1) using Primer Express software (Table ) and were ordered from IDT (USA). qRT-PCR was performed in an Applied Biosystems 7500 real-time PCR system under the following conditions: 50°C for 30 min, 95°C for 3 min, and 40 cycles of 94°C for 15 s and 60°C for 35 s. Negative template controls (NTCs) were used for each primer/probe set to exclude nonspecific reactions. No RT controls lacking Sensiscript reverse transcriptase were used to confirm the effective removal of genomic DNA. A standard curve was obtained for each primer/probe set using a serial 10-fold dilution of the growth control RNA sample (100–0.01 ng/reaction) to confirm the high dynamic range of their reaction efficiency (results not shown).The transcript FC, defined as the ratios of the mRNA expression level in the ciprofloxacin-exposed sample to that in the untreated sample, was calculated from the difference between the Ct values (as obtained from the 7500 Real-time PCR System Sequence Detection Software) of the ciprofloxacin-exposed samples (Ct treatment) and the Ct value of the untreated control sample (Ct control) using the equation: FC = 2−ΔCt (ΔCt = Ct treatment−Ct control).For RNA extracted from 0.5-ml culture volumes (where RNA concentrations were below the NanoDrop ND-1000 limit of detection), equal volumes (5 μl) of total RNA were used for the qRT-PCR, and the RNA concentrations were normalized using a 16S rRNA primer set (Table ) with a 1:1000 dilution of the samples. FC was calculated according to the ΔΔCt method (FC = 2−ΔΔCt where ΔΔCt = ΔCt marker gene−ΔCt16S RNA).Representative outcomes from two independent experiments are presented in the figures. […]

Pipeline specifications

Software tools Agilent Feature Extraction, limma, Primer Express, ddCt
Applications Gene expression microarray analysis, qPCR
Organisms Homo sapiens, Yersinia pestis
Diseases Yersinia Infections
Chemicals Ciprofloxacin