A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data

Background

Quantitative PCR (qPCR) is a workhorse laboratory technique for measuring the concentration of a target DNA sequence with high accuracy over a wide dynamic range. The gold standard method for estimating DNA concentrations via qPCR is quantification cycle () standard curve quantification, which requires the time- and labor-intensive construction of a standard curve. In theory, the shape of a qPCR data curve can be used to directly quantify DNA concentration by fitting a model to data; however, current empirical model-based quantification methods are not as reliable as standard curve quantification.

Principal Findings

We have developed a two-parameter mass action kinetic model of PCR (MAK2) that can be fitted to qPCR data in order to quantify target concentration from a single qPCR assay. To compare the accuracy of MAK2-fitting to other qPCR quantification methods, we have applied quantification methods to qPCR dilution series data generated in three independent laboratories using different target sequences. Quantification accuracy was assessed by analyzing the reliability of concentration predictions for targets at known concentrations. Our results indicate that quantification by MAK2-fitting is as reliable as standard curve quantification for a variety of DNA targets and a wide range of concentrations.

Significance

We anticipate that MAK2 quantification will have a profound effect on the way qPCR experiments are designed and analyzed. In particular, MAK2 enables accurate quantification of portable qPCR assays with limited sample throughput, where construction of a standard curve is impractical.     

Identification of a potentially neurotoxic pyridinium metabolite of haloperidol in rats

In vivo metabolic studies have revealed that haloperidol is converted to the corresponding pyridinium metabolite which has been characterized in both urine and brain tissues isolated from haloperidol treated rats. Unlike the corresponding conversion of the structurally related Parkinsonian inducing agent MPTP to the ultimate neurotoxic pyridinium metabolite MPP+, the oxidative biotransformation of haloperidol is not catalyzed by MAO-B. Microdialysis studies in the rat indicate that intrastriatal administration of this pyridinium metabolite is about 10% as effective as MPP+ in causing the irreversible depletion of striatal nerve terminal dopamine. The results point to the possibility that some of the neurological disorders observed in experimental animals and man during the course of chronic haloperidol treatment may be mediated by this pyridinium metabolite.     

Major histocompatibility (B) complex and sex effects on the phytohaemagglutinin wattle response

The influence of the major histocompatibility (B) complex and sex on the phytohaemagglutinin (PHA) wattle response was studied in 136 segregants (B2/B2, B2/B5 and B5/B5) of a fourth generation cross between inbred lines 6(1) and 15(1). At 6 weeks of age, chickens were injected with 100 micrograms purified PHA-P. Wattle thickness measurements were taken 4, 24, 48, 72 and 96 h after injection. Analysis of variance showed that 4 h after injection, males had a significantly higher response than females but the sex-genotype interaction was also significant. Females had higher responses than males 24 and 48 h after injection as a consequence of more rapid development and earlier resolution of the reaction in males. B2/B2 chickens had significantly lower responses than B5/B5 chickens 72 and 96 h after injection, signifying a faster late resolution phase in the B2/B2 genotype. The developmental and early resolution phases of the PHA wattle response were influenced by sex while the late resolution phase was influenced by B genotype.     

C : N : P stoichiometry of dominant riparian trees and arthropods along the Middle Rio Grande

1. We examined the role of flooding on the leaf nutrient content of riparian trees by comparing the carbon : nitrogen : phosphorus (C : N : P) ratio of leaves and litter of Rio Grande cottonwood (Populus deltoides ssp. wislizenii) in flood and non‐flood sites along the Middle Rio Grande, NM, U.S.A. The leaf C : N : P ratio was also examined for two non‐native trees, saltcedar (Tamarix chinensis) and Russian olive (Elaeagnus angustifolia), and six species of dominant riparian arthropods. 2. Living leaves and leaf litter of cottonwoods at flood sites had a significantly lower leaf N : P ratio and higher %P compared with leaves and litter at non‐flood sites. A non‐flood site downstream from wastewater effluent had a significantly lower litter C : N ratio than all other sites, suggesting N fertilisation through ground water. The non‐native trees, saltcedar and Russian olive, had higher mean leaf N content, N : P ratio, and lower C : N ratio compared with cottonwoods across study sites. 3. Riparian arthropods ranged from 5.2 to 7.1 for C : N ratio, 56–216 for C : P ratio, and 8.9–34 for N : P ratio. C content ranged from 25 to 52% of dry mass, N content from 4.7 to 10.8%, and P content from 0.59 to 1.2%. Differences in stoichiometry between high C : nutrient leaf litter and low C : nutrient invertebrates suggests possible food‐quality constraints for detritivores. 4. These results suggest that spatial and temporal variation in the C : N : P ratio of cottonwood leaves and leaf litter is influenced by surface and subsurface hydrologic connection within the floodplain. Reach‐scale variation in the elemental composition of riparian organic matter inputs may have important implications for decomposition, nutrient cycling, and food webs in river floodplain systems.