We present a Bayesian hierarchical model for quantitative real-time polymerase chain reaction (PCR) data, aiming at relative quantification of DNA copy number in different biological samples. The model is specified in terms of a hidden Markov model for fluorescence intensities measured at successive cycles of the polymerase chain reaction. The efficiency of the reaction is assumed to depend on ...

Quantitative lytA PCR is often performed using in-house standards. We hypothesised equivalence when measuring a standard suspension of Streptococcus pneumoniae by colony-forming-units (CFU) or genome-copies. Median (IQR) ratio of CFU/genome-copies was 0.19 (0.1-1.2). Genome-copies were less variable than CFU, but the discrepancy between the methods highlights challenges with absolute quantifica...

Helicobacter pylori is one of the most common chronic infections in humans, in whom it is a key etiological factor in peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. Humans are the bacterium's only host. Here we report the development of a real-time quantitative (Q) PCR-based assay to measure ureC gene copy number to detect H. pylori, based ...

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6...

Gene expression studies employing high throughput real time PCR methods require finding uniform conditions for optimal amplification of multiple targets, often a daunting task. We developed a primer database, qPrimerDepot, which provides optimized primers for all human and mouse RefSeq genes. These primers are designed to amplify desired templates under unified annealing temperature. For most i...

We have utilized an in vitro transcribed 3' mRNA fragment of the plant gene ribulose bisphosphate carboxylase (RuBisCO) as an exogenous standard for normalization of quantitative PCR data. Both K562 cells and primary erythroid CD34+ progenitor cells were treated with sodium butyrate and changes in gamma-globin mRNA levels were assayed using a previously published TaqMan probe and primer set, wh...

Vol. 35, No. 6 (2003) BioTechniques 1141 the ABI PRISM® 7000 and the TaqMan Universal PCR Master Mix protocols (Applied Biosystems). After discovering that the RNA from the thecal cells had vascular endothelial growth factor (VEGF) amplification and that RNA from granulosa cells had no VEGF amplification, the 18S rRNA amplification (TaqMan Pre-Developed Assay Reagents; Applied Biosystems), whic...

Over the past decade, PCR-based methodologies have been introduced to complement or even replace histopathologic study of biopsy specimens for the diagnosis of Whipple’s disease (12). However, positive PCR results have been reported on testing small-bowel and saliva specimens from asymptomatic patients (3, 4, 11). Although these results have not been independently confirmed (8), they have nonet...

Waterborne diseases represent a significant public health risk worldwide and can originate from contact with water contaminated with human fecal material. We describe a real-time quantitative PCR (qPCR) method that targets a genetic marker of the human-associated Bacteroides dorei for identification of human fecal pollution in ambient water samples. The following protocol includes water sample ...