Submitted to: Weed Science Society of America Meeting Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 10/1/2006
Publication Date: 1/13/2007
Citation: Chao, W.S. 2007. Real-time PCR as a tool to study weed biology. [Abstract] Weed Science Society of America. Presentation No. 147.
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 round of amplification throughout PCR cycles. Additionally, it does not require running gels and thus is able to produce data in 2-3 h. Four different chemistries, Taqman, Molecular Beacons, Scorpions, and SYBR green, have been used for Real-time PCR. Among these chemistries, SYBR green is the most economical choice. We have applied 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 control genes have been identified for growing bud samples in leafy spurge, we performed PCR without endogenous controls and analyzed mRNA expression semi-quantitatively using the raw fluorescence data collected during the exponential phase of PCR. Our results indicated that excluding endogenous controls in data analysis was straightforward and reliable if 1) RNA was properly quantified using Nanodrop instrumentation, and 2) the purpose of the experiment was simply to determine an increase or decrease in target template. Given that genomic tools such as ESTs and their expression profiles are lacking for most weedy species, avoiding the use of endogenous controls in Real-time PCR simplifies the optimization process and reduces the cost tremendously. We also found that using a passive reference dye (ROX) to normalize non-PCR-related fluctuations in fluorescence signal is desirable.