SYBR Green Based Real Time PCR For Gene Expression Studies In Salmonella Enterica

August 04, 2016

University of Birmingham
Biosciences
Research Assistant

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Company:

Bioline

Product Name:

SensiFAST SYBR Lo-Rox Kit

Catalog Number:

BIO-94002

I used SensiFAST Lo-Rox Kit for measuring Salmonella enterica gene expression under bile salt stress. The standard curve analysis using SensiFAST SYBR Lo Rox kit was 99% efficient. Assay performed well and was able to relatively quantify genes expressed during bile stress. Gene expression values (Cqs) for three biological replicates were consistent with less deviation among the replicates.

Experimental Design and Results Summary

Application

SYBR Green based Real Time PCR experiments to measure gene expression

Starting Material

Salmonella enterica cDNA prepared from Total RNA extracted under bile salt stress

Protocol Overview

All reactions (samples and standards) prepared in a 20 microliter reaction using 10 microliter of 2X SenisFAST SYBR Lo-Rox master mix along with primer pairs for selected targets at 500 nM final concentration. cDNA was diluted in 1:10 and 2 microliter of this diluted cDNA was used as template in the reaction. For generation of standard curve, 10 fold diluted (up to 6 point dilution) genomic DNA was used. Cycling conditions were performed as per the kit's instructions. Relative quantification of genes expressed during bile stress were analysed using internal reference gene.

Tips

Protect SensiFAST SYBR Lo Rox kit from light exposure during storage.

Results Summary

Standard curve analysis revealed 99% efficiency of the reaction. Relative quantification of genes expected to be expressed during the bile stress in Salmonella enterica among three biological replicates were consistent with less deviation. Total satisfactory relative quantification were seen.

Additional Notes

None

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Summary

The Good

Easy to use protocol. Less expensive kit compared to Agilent SYBR green based kits.

The Bad

None

The Bottom Line

If you are routinely doing gene expression quantification (relative) using SYBR green based chemistry, then go for Bioline's SensiFAST Lo-Rox kit since it slightly less expensive than Agilent kits.

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