Author
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Chao, Wun |
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Submitted to: Weed Science
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 6/27/2007 Publication Date: 3/1/2008 Citation: Chao, W.S. 2008. Real-time PCR as a tool to study weed biology. Weed Science. 56:290-296. Interpretive Summary: Real-time polymerase chain reaction (real-time PCR) also known as quantitative PCR is used to determine relative gene expression or to quantify exact levels of mRNA in cells or tissues. Before the advent of real-time PCR, the major difficulty associated with traditional quantitative or semi-quantitative PCR was to ensure that PCR reactions were quantified within the linear range of amplification. Real-time PCR alleviates this problem since it detects and quantifies fluorescent signal after each amplification cycle. Additionally, it does not require running gels and thus is able to produce data in 2-3 h. Four different types of chemistries, DNA-binding agents (SYBR Green), hydrolysis probes (TaqMan), hairpin probes (molecular beacons, scorpions), and fluorescent-labelled hybridization probes (Light Cycler) have been commonly used for real-time PCR. Among these chemistries, SYBR green is the most economical choice. We have used real-time PCR and SYBR green to examine the expression of a number of leafy spurge genes after growth induction and during normal seasonal growth. Since no reliable endogenous reference genes have been identified in leafy spurge, we performed PCR without endogenous reference genes and analyzed mRNA expression based on the CT value of amplification. Excluding endogenous reference genes from this data analysis was rather straightforward and reliable if RNA was properly prepared and quantified. Given that genomic tools such as ESTs and their expression profiles are lacking for most weedy species, avoiding the use of endogenous reference genes in real-time PCR simplifies the optimization process and reduces the cost tremendously. Technical Abstract: Real-time polymerase chain reaction (real-time PCR) also known as quantitative PCR is used to determine relative gene expression or to quantify exact levels of mRNA in cells or tissues. Before the advent of real-time PCR, the major difficulty associated with traditional quantitative or semi-quantitative PCR was to ensure that PCR reactions were quantified within the linear range of amplification. Real-time PCR alleviates this problem since it detects and quantifies fluorescent signal after each amplification cycle. Additionally, it does not require running gels and thus is able to produce data in 2-3 h. Four different types of chemistries, DNA-binding agents (SYBR Green), hydrolysis probes (TaqMan), hairpin probes (molecular beacons, scorpions), and fluorescent-labelled hybridization probes (Light Cycler) have been commonly used for real-time PCR. Among these chemistries, SYBR green is the most economical choice. We have used real-time PCR and SYBR green to examine the expression of a number of leafy spurge genes after growth induction and during normal seasonal growth. Since no reliable endogenous reference genes have been identified in leafy spurge, we performed PCR without endogenous reference genes and analyzed mRNA expression based on the CT value of amplification. Excluding endogenous reference genes from this data analysis was rather straightforward and reliable if RNA was properly prepared and quantified. Given that genomic tools such as ESTs and their expression profiles are lacking for most weedy species, avoiding the use of endogenous reference genes in real-time PCR simplifies the optimization process and reduces the cost tremendously. However, we found that using a passive reference dye (ROX) to normalize non-PCR-related fluctuations in fluorescence signal is desirable. |
