Quantitative Transcriptional Profiling of Chlamydial inc Genes

Quantitative transcriptional assays for the indicated inc genes were performed as described previously (Ouellette et al., 2014b (link)). Briefly, total RNA was collected from C. trachomatis L2 infected HeLa cells at the indicated times using Trizol (Invitrogen) and treated with Turbo DNAfree (Ambion, Life Technologies) to remove contaminating DNA, according to the manufacturer's guidelines. One μg DNA-free RNA was reverse-transcribed with random nonamers (New England Biolabs, Ipswich, MA) using SuperScript III RT (Invitrogen) according to the manufacturer's instructions. Equal volumes of cDNA were used in qPCR reactions with SYBR Green (Quanta Biosciences, Gaithersburg, MD) and measured on an ABI 7300 system (Applied Biosystems, Life Technologies). Duplicate DNA samples were collected from the same experiment using Dneasy Tissue kit (Qiagen). Chlamydial genomes were quantified from equal amounts of total DNA by qPCR as above and used to normalize transcript data as described (Ouellette et al., 2005 (link), 2006 (link)).

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Gauliard E., Ouellette S.P., Rueden K.J, & Ladant D. (2015). Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis. Frontiers in Cellular and Infection Microbiology, 5, 13.

Publication 2015

Trizol Turbo DNAfree SuperScript III RT SYBR Green ABI 7300 system Dneasy Tissue kit

Assays Cdna Chlamydial Genes Genomes Hela cells Sybr green Tissue Transcriptional Trizol

Corresponding Organization : University of South Dakota

Top 5 similar protocols

Variable analysis

independent variables

Infection of HeLa cells with C. trachomatis L2

dependent variables

Quantitative transcriptional levels of the indicated inc genes

control variables

Time points for RNA collection from C. trachomatis L2 infected HeLa cells
Extraction of total RNA using Trizol
DNase treatment of RNA to remove contaminating DNA
Reverse transcription of 1 μg DNA-free RNA using random nonamers and SuperScript III RT
Use of equal volumes of cDNA in qPCR reactions with SYBR Green
Measurement of qPCR on an ABI 7300 system
Collection of duplicate DNA samples from the same experiment using Dneasy Tissue kit
Quantification of chlamydial genomes from equal amounts of total DNA by qPCR and use of this to normalize transcript data

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