用于分析金黄色葡萄球菌基因表达的定量实时PCR（qPCR）工作流程。

Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.

作者信息

Lewis April M, Rice Kelly C

机构信息

Department of Microbiology and Cell Science, IFAS, University of Florida, Gainesville, FL, 32611-0700, USA.

出版信息

Methods Mol Biol. 2016;1373:143-54. doi: 10.1007/7651_2014_193.

DOI:10.1007/7651_2014_193

PMID:25646613

Abstract

Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a "two-step" qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specific genes of interest using SYBR Green-based qPCR and quantification of relative fold-change expression.

摘要

定量实时聚合酶链反应（qPCR）是一种灵敏的工具，可用于定量和比较不同生物样品之间特定RNA转录本的数量。本章介绍了使用“两步法”qPCR方法来计算金黄色葡萄球菌中感兴趣基因表达的相对倍数变化。使用该工作流程，利用随机引物和逆转录酶的混合物，从RNA模板（先前已检查是否不存在显著的基因组DNA污染）合成cDNA。然后，可以使用基于SYBR Green的qPCR评估生成的cDNA池，以分析特定感兴趣基因的表达，并对相对倍数变化表达进行定量。