Relative Quantification of Bacterial Gene Expression

March 14, 2016

University of Birmingham
School of Biosciences
Postdoctoral Research Fellow

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Company:

Agilent Technologies Inc

Product Name:

Brilliant III Ultra-Fast SYBR Green qPCR Master mix

Catalog Number:

600882

Expression of Escherichia coli genes responsible for acid resistance were measured in relative terms using Agilent's brilliant ultra-fast SYBR green qPCR master mix in a 20 microliter reaction. E. coli cells exposed to acid stress had significantly higher expression of acid resistance genes (gadA and CadA) compared to control cells exposed to neutral pH. Agilent's SYBR green based ultra-fast qPCR master mix gives reliable quantification of genes in relative expression terms.

Experimental Design and Results Summary

Application

Relative quantification of bacterial gene expression

Starting Material

cDNA synthesized from E.coli cells exposed to acidic and neutral pH in a LB broth

Protocol Overview

cDNA were synthesized from total RNA extracted from E. coli cells grown under acidic and neutral pH. cDNAs were diluted into 1:5 in a nuclease free water. qPCR was performed using 10 microliter of Brilliant III ultra-fast SYBR green qPCR master mix , 4 microliter of cDNA as template and 500 nM primers in a 20 microliter reaction volume according to manufacturer's protocol in Agilent's MX3500P suit. Standard curves were established using known DNA standards. Expression of genes were quantified in relative terms using csrA as a normalizer.

Tips

Proper designing of primers to amplify your gene interest is critical for successful qPCR quantification especially avoid formation of secondary structures in your design

Results Summary

Relative quantification of acid response genes in the E. coli cells grown in acidic condition was found to be significantly higher than control cells grown in neutral pH.

Additional Notes

None

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Summary

The Good

Less expensive and easy perform assay compared to other probe based metehods

The Bad

Specificity is less compared to TaqMan probe based assays

The Bottom Line

Ideal assay kit for relative quantification of large number of genes in your study with proper care taken during primer design. Less expensive.

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