Accurate and Efficient Data Processing for Quantitative Real-Time PCR using a Tripartite Plant Virus as a Model

Abstract

Real-time PCR is becoming a preferred method for quantification of minute amounts of nucleic acids. To achieve the full potential of this technique, accurate and convenient models for post-PCR data analysis are required. In this study, three different models were chosen to quantify the definitive copy numbers of Cucumber mosaic virus (CMV) genomic RNAs using raw fluorescence data of real-time PCR, and equations were proposed to compare their expression levels in virions or in planta. The results, as confirmed by standard curve and Northern blotting methods, show that the expression levels of different genes can be compared more accurately and more efficiently by these equations, especially using theoretical fluorescence (F0) and calibration factors (CF), determined by linear regression PCR (LinRegPCR). Thus, these equations, combined with data analysis by the LinRegPCR method, can greatly enhance the high-throughput quantification ability of real-time PCR, and permit accurate, reliable, and facile investigation of the changes in CMV RNAs accumulation.

Gene copy number is differentially regulated in a multipartite virus

“…Although no direct investigation has addressed this question, some data indicating unequal accumulation of different genome segments can be found, for example, in studies on cucumber mosaic virus [38][39][40][41] , tomato aspermy virus 42,43 and brome mosaic virus 44 -all positive-sense ssRNA viruses unrelated to FBNSV. These earlier studies were either purely descriptive, analysing the physical and/or chemical properties of the virus particle 38,39,42,44 , or focused on technical developments for RNA quantification 40,41,44 . Nevertheless, they suggest that the regulation of segment copy number may be a general feature of multipartite viruses.…”

Defects in RNA polyadenylation impair both lysogenization by and lytic development of Shiga toxin-converting bacteriophages

“…PCR efficiency was determined for each gene by LinRegPCR program (Ramakers et al, 2003;Ruijter et al, 2009). This software was successfully used previously to calculate PCR efficiency (Č ikoš et al, 2007;Feng et al, 2008;Regier & Frey, 2010;Aglawe et al, 2012, Borges et al, 2012.…”

Spatio-temporal expression of miRNAs in tomato tissues upon <italic>Cucumber mosaic virus</italic> and <italic>Tomato aspermy virus</italic> infections

“…Immediately after the final PCR cycle, a melting curve analysis was done to determine the specificity of each reaction. The threshold cycle (C T ) values were determined automatically by the instrument, and the fold changes of each gene were calculated as relative quantity (RQ) values using the comparative C T (2 2DDCt ) method [10,17].…”