ArticleExpert review of molecular diagnostics2015
Properties of targeted preamplification in DNA and cDNA quantification.
Article in Expert review of molecular diagnostics, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 27 papers.
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Who cites it
27 citing papers in PubMed, 51 citations in OpenAlex.
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- Absolute Quantification of Donor-Derived Cell-Free DNA in Pediatric and Adult Patients After Heart Transplantation: A Prospective Study.Transplant international : official journal of the European Society for Organ Transplantation · 2023Observational
- Extracellular vesicle-bound DNA in urine is indicative of kidney allograft injury.Journal of extracellular vesicles · 2022Article
- Rapid and highly sensitive approach for multiplexed somatic fusion detection.Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc · 2022Article
- Eight weeks of combined exercise training do not alter circulating microRNAs-29a, -133a, -133b, and -155 in young, healthy men.European journal of applied physiology · 2022Article
- Laser capture microdissection to study Bacillus cereus iron homeostasis gene expression during Galleria mellonella in vivo gut colonization.Virulence · 2021Article
- Tutorial: Guidelines for Single-Cell RT-qPCR.Cells · 2021Review
- Highly sensitive and multiplexed one-step RT-qPCR for profiling genes involved in the circadian rhythm using microparticles.Scientific reports · 2021Article
- Validation of Preamplification to Improve Quantification of Cytomegalovirus DNA Using Droplet Digital Polymerase Chain Reaction.Analytical chemistry · 2021Article
- Pre-amplification as a method for improvement of quantitative RT-PCR analysis of circulating miRNAs.Biochemia medica · 2021Article
- Non-Invasive Diagnosis for Acute Rejection Using Urinary mRNA Signature Reflecting Allograft Status in Kidney Transplantation.Frontiers in immunology · 2021Article
- Impact of Polymerase Fidelity on Background Error Rates in Next-Generation Sequencing with Unique Molecular Identifiers/Barcodes.Scientific reports · 2019Article
- Challenging the proposed causes of the PCR plateau phase.Biomolecular detection and quantification · 2019Article
- Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons.International journal of molecular sciences · 2018Article
Corrections and comments
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Authors and funding
7 authors at 4 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
objectiveQuantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR).
methodsTo evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR.
resultThe preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification.
conclusionOn the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.