Application of the common base method to regression and analysis of covariance (ANCOVA) in qPCR experiments and subsequent relative expression calculation.

BACKGROUND: Quantitative polymerase chain reaction (qPCR) is the technique of choice for quantifying gene expression. While the technique itself is well established, approaches for the analysis of qPCR data continue to improve. RESULTS: Here we expand on the common base method to develop procedures for testing linear relationships between gene expression and either a measured dependent variable, independent variable, or expression of another gene. We further develop functions relating variables to a relative expression value and develop calculations for determination of associated confidence intervals. CONCLUSIONS: Traditional qPCR analysis methods typically rely on paired designs. The common base method does not require such pairing of samples. It is therefore applicable to other designs within the general linear model such as linear regression and analysis of covariance. The methodology presented here is also simple enough to be performed using basic spreadsheet software.

A soil bacterium alters sex determination and rhizoid development in gametophytes of the fern Ceratopteris richardii.

Gametophytes of the fern Ceratopteris richardii develop into either hermaphrodites or males. As hermaphrodites develop, they secrete antheridiogen, or ACE, into the environment, inducing male development in undifferentiated gametophytes. Hermaphrodites are composed of archegonia, antheridia, rhizoids and a notch meristem, while males consist of antheridia and rhizoids. Much of the research on sexual and morphological development concerns gametophytes grown in sterile environments. Using biochemical and molecular techniques we identify a soil bacterium and explore its effects on sexual and rhizoid development. Hermaphrodite and male gametophytes were exposed to this bacterium and the effects on sexual development, rhizoid length and rhizoid number were explored. The bacterium was identified as a pseudomonad, Pseudomonas nitroreducens. Gametophytes grown in the presence of the pseudomonad were more likely to develop into hermaphrodites across all gametophyte densities. Across all gametophyte sizes, hermaphrodites had rhizoids that were 2.95× longer in the presence of the pseudomonad while males had rhizoids that were 2.72× longer in the presence of the pseudomonad. Both hermaphrodite and male gametophytes developed fewer rhizoids in the presence of the pseudomonad. Control hermaphrodites produced 1.23× more rhizoids across all gametophyte sizes. For male gametophytes grown in the absence of the pseudomonad, the rate of increase in the number of rhizoids was greater with increasing size in the control than the rate of increase in males grown in the presence of the pseudomonad. The pseudomonad may be acting on gametophyte sexual development via several potential mechanisms: degradation of ACE, changes in nutrient availability or phytohormone production. The pseudomonad may also increase rhizoid number through production of phytohormones or changes in nutrient availability.

A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments.

BACKGROUND: qPCR has established itself as the technique of choice for the quantification of gene expression. Procedures for conducting qPCR have received significant attention; however, more rigorous approaches to the statistical analysis of qPCR data are needed. RESULTS: Here we develop a mathematical model, termed the Common Base Method, for analysis of qPCR data based on threshold cycle values (C q ) and efficiencies of reactions (E). The Common Base Method keeps all calculations in the logscale as long as possible by working with log10(E) ∙ C q , which we call the efficiency-weighted C q value; subsequent statistical analyses are then applied in the logscale. We show how efficiency-weighted C q values may be analyzed using a simple paired or unpaired experimental design and develop blocking methods to help reduce unexplained variation. CONCLUSIONS: The Common Base Method has several advantages. It allows for the incorporation of well-specific efficiencies and multiple reference genes. The method does not necessitate the pairing of samples that must be performed using traditional analysis methods in order to calculate relative expression ratios. Our method is also simple enough to be implemented in any spreadsheet or statistical software without additional scripts or proprietary components.

The influence of pollen addition and ramet isolation on current sexual reproduction in a clonal herb.

Canada mayflower (Maianthemum canadense Desf.), a rhizomatous, perennial herb, was the subject of a 2-year field experiment that examined two factors potentially affecting fruit and seed production: pollen addition and ramet isolation. Ramets were either open pollinated or overpollinated by hand to supplement natural levels. Rhizomes of the ramets were either severed, to prevent resource supplementation from the genet, or left intact. Ramets that were overpollinated matured more fruits and more seeds than ramets that were open pollinated. Thus, mayflower appears to have been pollen limited in both years. Ramets that were open pollinated and whose rhizomes were severed matured as many fruits, seeds, and seeds/fruit as ramets that were open pollinated and whose rhizomes were left intact. Ramets that were overpollinated and whose rhizomes were severed matured fewer fruits, seeds, and seeds/fruit than overpollinated ramets whose rhizomes were left intact. It appears that at natural levels of pollination, mayflower ramets are physiologically independent but as the level of pollen increases, mayflower ramets receive support from other parts of the genet.