Analyzing Staphylococcus aureus Virulence Genes

Bacterial RNA was extracted from S. aureus grown in TSB until post-exponential phase, using TRIzol Reagent (Invitrogen Life Technologies), according to the manufacturer’s protocol. RNA was subjected to DNAse treatment using a RQ1 RNAse free DNAse (Promega). cDNA synthesis was performed with an ImProm-II Reverse Transcriptase kit (Promega). qRT-PCR for RNAIII, agrA and asp23 expression were performed using the primers and conditions described in a previous publication^{9 (link)} and the SYBR Green PCR Master Mix (Applied Biosystems) equipment and kits. The 16S gene was used to normalize data. The (− ΔCt) value represents the difference in threshold cycle (Ct) between the target and control(16S) genes^{65 (link)}.

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Suligoy C.M., Díaz R.E., Gehrke A.K., Ring N., Yebra G., Alves J., Gómez M.I., Wendler S., FITZGERALD J.R., Tuchscherr L., Löffler B., Sordelli D.O., Llana M.N, & Buzzola F.R. (2020). Acapsular Staphylococcus aureus with a non-functional agr regains capsule expression after passage through the bloodstream in a bacteremia mouse model. Scientific Reports, 10, 14108.

Publication 2020

TRIzol Reagent RQ1 RNAse free DNAse ImProm-II Reverse Transcriptase kit SYBR Green PCR Master Mix

Bacterial rna Cdna Dnase Gene Primers Promega Reverse transcriptase Rnaiii Rnase Sybr green Synthesis Trizol

Corresponding Organization :

Other organizations : Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Maimónides, Roslin Institute, University of Edinburgh, Jena University Hospital

Top 5 similar protocols

Variable analysis

independent variables

Growth phase of S. aureus (post-exponential)

dependent variables

Expression of RNAIII
Expression of agrA
Expression of asp23

control variables

Bacterial strain (S. aureus)
Culture medium (TSB)
RNA extraction method (TRIzol Reagent)
DNAse treatment (RQ1 RNAse free DNAse)
CDNA synthesis (ImProm-II Reverse Transcriptase kit)
QRT-PCR method (SYBR Green PCR Master Mix)
Normalization gene (16S)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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