塔克曼
底漆(化妆品)
互补DNA
逆转录酶
生物
生物膜
逆转录聚合酶链式反应
分子生物学
基因表达
实时聚合酶链反应
基因
激光捕获显微切割
计算生物学
聚合酶链反应
细菌
化学
遗传学
有机化学
作者
Ailyn C. Pérez-Osorio,Michael J. Franklin
出处
期刊:CSH Protocols
[Cold Spring Harbor Laboratory]
日期:2008-10-01
卷期号:2008 (10): pdb.prot5066-pdb.prot5066
被引量:8
摘要
INTRODUCTION Bacteria growing in biofilms often express a different subset of genes compared to the same strains growing planktonically. Quantitative reverse transcriptase real time PCR (qRT-PCR) can be used effectively to quantify the number of RNA transcripts of specific genes from bacteria growing in biofilms. qRT-PCR has a large dynamic range and may be used to verify gene expression data obtained from microarrays. In addition, qRT-PCR is sensitive, and therefore may be used to quantify gene expression from biofilm samples where only a small amount of biological material is available, as in samples obtained by laser capture microdissection microscopy (LCMM). The most commonly used qRT-PCR methods are the SYBR Green and dual-labeled probe (Taqman) approaches. Both approaches use reverse transcription to convert mRNA to cDNA, followed by PCR amplification of the cDNA. This article describes steps involved in aspects of qRT-PCR including (1) primer design, (2) primer and probe performance testing, (3) qRT-PCR using the Corbett Rotor-Gene system, and (4) data export and analysis.
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