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Determination and Quantification of Bacterial Virulent Gene Expression Using Quantitative Real-Time PCR

  • Qiao Lin
  • Y. Peter DiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2102)

Abstract

Polymerase chain reaction (PCR) plays significant roles in modern molecular biology. However, it is relatively cumbersome and less accurate to use the traditional PCR method in quantifying gene expression because it requires first generating a standard curve with multiple input controls showing linearity with amplified control PCR products on a electrophoresis gel to compare with the abundance of the to-be-determined gene transcript PCR amplicons. Quantitative real-time PCR (qRT-PCR) is a time-efficient and reliable tool for accurate quantification and comparison of gene (RNA transcript) expression from various biological samples. Current technology has simplified and expedited the qPCR process significantly. However, proper techniques and standard protocols are required in eliminating potentially erroneous experimental outcome. Here, we provide an example from a drug-treated bacterial gene expression study with detailed protocols to demonstrate real-time qPCR with SYBR Green and TaqMan®, two of the most adapted and well-established qPCR technologies. Relative quantification of gene (RNA transcript) expression using qRT-PCR is demonstrated in detail from sample preparations to data analysis.

Key words

qPCR SYBR Green TaqMan Real-time PCR Gene expression 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Environmental and Occupational HealthUniversity of PittsburghPittsburghUSA

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