Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability.

Chelsey Hilscher; Wolfgang Vahrson; Dirk P Dittmer

doi:10.1093/nar/gni181

Home/Archive/PubMed

PubMed · 16314296

Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability.

Chelsey Hilscher·Wolfgang Vahrson·Dirk P Dittmer

View original source ↗Export metadata ↓

Abstract

Quantitative real-time PCR has become the method of choice for measuring mRNA transcription. Recently, fast PCR protocols have been developed as a means to increase assay throughput. Yet it is unclear whether more rapid cycling conditions preserve the original assay performance characteristics. We compared 16 primer sets directed against Epstein-Barr virus (EBV) mRNAs using universal and fast PCR cycling conditions. These primers are of clinical relevance, since they can be used to monitor viral oncogene and drug-resistance gene expression in transplant patients and EBV-associated cancers. While none of the primers failed under fast PCR conditions, the fast PCR protocols performed worse than universal cycling conditions. Fast PCR was associated with a loss of sensitivity as well as higher variability, but not with a loss of specificity or with a higher false positive rate.

Explore related subjects

DNA Primers Herpesvirus 4, Human Molecular Diagnostic Techniques Polymerase Chain Reaction RNA, Messenger Reproducibility of Results Sensitivity and Specificity Time Factors Transcription, Genetic Comparative Study Evaluation Study Journal Article Research Support, N.I.H., Extramural

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Chelsey Hilscher, Wolfgang Vahrson, Dirk P Dittmer. 2005-11-27. Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability.. https://doi.org/10.1093/nar%2Fgni181

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2025

AutoPVPrimer: A comprehensive AI-Enhanced pipeline for efficient plant virus primer design and assessment.

Plant viruses pose a significant threat to global agriculture and require efficient tools for their timely detection. We present AutoPVPrimer, an innovative pipeline that integrates artificial intelligence (AI) and machine learning to accelerate the development of plant virus primers. The pipeline uses Biopython to automatically retrieve different genomic sequences from the NCBI database to increase the robustness of the subsequent primer design. The design_primers_with_tuning module uses a random forest classifier that optimizes parameters and provides flexibility for different experimental conditions. Quality control measures, including the evaluation of poly-X content and melting temperature, increase primer reliability. Unique to AutoPVPrimer is the visualize_primer_dimer module, which supports the visual evaluation of primer dimers-a feature missing in other tools. Primer specificity is validated via primer BLAST, which contributes to the overall efficiency of the pipeline. AutoPVPrimer has been successfully applied to the tomato mosaic virus, proving its adaptability and efficiency. The modular design allows customization by the user and extends the applicability to different plant viruses and experimental scenarios. The pipeline represents a significant advance in primer design and provides researchers with an effective tool to accelerate molecular biology experiments. Future developments aim to extend compatibility and incorporate user feedback to consolidate AutoPVPrimer as an innovative contribution to the bioinformatics toolbox and a promising resource for the advancement of plant virology research.

DNA Primers↗

PubMed2007

Global genome expression analysis of rice in response to drought and high-salinity stresses in shoot, flag leaf, and panicle.

To elucidate genome-level responses to drought and high-salinity stress in rice, a 70 mer oligomer microarray covering 36,926 unique genes or gene models was used to profile genome expression changes in rice shoot, flag leaf and panicle under drought or high-salinity conditions. While patterns of gene expression in response to drought or high-salinity stress within a particular organ type showed significant overlap, comparison of expression profiles among different organs showed largely organ-specific patterns of regulation. Moreover, both stresses appear to alter the expression patterns of a significant number of genes involved in transcription and cell signaling in a largely organ-specific manner. The promoter regions of genes induced by both stresses or induced by one stress in more than one organ types possess relative enrichment of two cis-elements (ABRE core and DRE core) known to be associated with water stress. An initial computational analysis indicated that novel promoter motifs are present in the promoters of genes involved in rehydration after drought. This analysis suggested that rice might possess a mechanism that actively detects rehydration and facilitates rapid recovery. Overall, our data supports a notion that organ-specific gene regulation in response to the two abiotic stresses may primarily be mediated by organ-specific transcription responses.

DNA Primers↗

PubMed2007

Genotyping of single-nucleotide polymorphisms by primer extension reaction in a dry-reagent dipstick format.

The primer extension (PEXT) reaction is the most widely used approach to genotyping of single-nucleotide polymorphisms (SNPs). Current methods for analysis of PEXT reaction products are based on electrophoresis, fluorescence resonance energy transfer, fluorescence polarization, pyrosequencing, mass spectrometry, microarrays, and spectrally encoded microspheres. We report the first dry-reagent dipstick method that enables rapid visual detection of PEXT products without instrumentation. The method is applied to the analysis of six SNPs in the mannose-binding lectin gene (MBL2). The genomic region that spans each SNP of interest is amplified by PCR. Two primer extension reactions are performed with allele-specific primers (for one or the other variant nucleotide), which contain an oligo(dA) segment at the 5'-end. Biotin-dUTP is incorporated in the extended strand. The product is applied to the strip followed by immersion in the appropriate buffer. As the DNA moves along the strip by capillary action, it hybridizes with oligo(dT)-functionalized gold nanoparticles, such that only extended products are captured by immobilized streptavidin at the test zone, generating a red line. A second red line is formed at the control zone of the strip by hybridization of the nanoparticles with immobilized oligo(dA). The dipstick test is complete within 10 min. We analyzed six SNPs of the mannose-binding lectin gene (MBL2) using genomic DNA from 27 patients, representing a total of 74 variant nucleotide positions. Patient genotypes showed 100% concordance with direct DNA sequencing data. The described PEXT-dipstick assay is rapid and highly accurate; it does not require specialized instrumentation or highly trained technical personnel. It is appropriate for a diagnostic laboratory where a few selected SNP markers are examined per patient with a low cost per assay.

DNA Primers↗