Abstract:
BACKGROUND:The challenge of determining amplification efficiency has long been a predominant aspect of implementing real-time qPCR, playing a critical role in the accuracy and reliability that can be achieved. Based upon analysis of amplification profile position, standard curves are currently the gold standard for amplification efficiency determination. However, in addition to being highly resource intensive, the efficacy of this approach is limited by the necessary assumption that all samples are amplified with the same efficiency as predicted by a standard curve. These limitations have driven efforts to develop methods for determining amplification efficiency by analyzing the fluorescence readings from individual amplification reactions. The most prominent approach is based on analysis of the "log-linear region", founded upon the presumption that amplification efficiency is constant within this region. Nevertheless, a recently developed sigmoidal model has provided new insights that challenge such historically held views, dictating that amplification efficiency is not only dynamic, but is linearly coupled to amplicon DNA quantity. Called "linear regression of efficiency" or LRE, this kinetic-based approach redefines amplification efficiency as the maximal efficiency (Emax) generated at the onset of thermocycling. RESULTS:This study presents a critical evaluation of amplification efficiency determination, which reveals that potentially large underestimations occur when exponential mathematics is applied to the log-linear region. This discrepancy was found to stem from misinterpreting the origin of the log-linear region, which is derived not from an invariant amplification efficiency, but rather from an exponential loss in amplification rate. In contrast, LRE analysis generated Emax estimates that correlated closely to that derived from a standard curve, despite the fact that standard curve analysis is founded upon exponential mathematics. This paradoxical result implies that the quantitative efficacy of positional-based analysis relies not upon the exponential character of real-time PCR, but instead on the ability to precisely define the relative position of an amplification profile. CONCLUSION:In addition to presenting insights into the sigmoidal character of the polymerase chain reaction, LRE analysis provides a viable alternative to standard curves for amplification efficiency determination, based on analysis of high-quality fluorescence readings within the central region of SYBR Green I generated amplification profiles.
journal_name
BMC Mol Bioljournal_title
BMC molecular biologyauthors
Rutledge RG,Stewart Ddoi
10.1186/1471-2199-9-96subject
Has Abstractpub_date
2008-10-30 00:00:00pages
96issn
1471-2199pii
1471-2199-9-96journal_volume
9pub_type
杂志文章abstract:BACKGROUND:The human Sex Hormone Binding Globulin (SHBG) gene, located at 17p13.1, comprises, at least, two different transcription units regulated by two different promoters. The first transcription unit begins with the exon 1 sequence and is responsible for the production of plasma SHBG by the hepatocytes, while the ...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-10-59
更新日期:2009-06-17 00:00:00
abstract:BACKGROUND:Senescence marker protein-30 (SMP30), whose expression declines during aging in rat liver, has been proposed as an important aging marker. Besides apoptosis, SMP30 also protects cells against various other injuries by enhancement of membrane calcium-pump activity. The mechanism of this differential gene expr...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-9-43
更新日期:2008-04-29 00:00:00
abstract:BACKGROUND:RA (retinoic acid) signal pathway has been proved to be required for germ cell meiotic initiation in mammals, aves and amphibians. Stra8 (Stimulated by retinoic acid gene 8) is an important factor in RA signal pathway. However, the role of RA and Stra8 in germ cell meiotic initiation in teleosts is poorly ch...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-14-11
更新日期:2013-05-22 00:00:00
abstract:BACKGROUND:Double-strand breakage of chromosomal DNA is obviously a serious threat to cells because various activities of the chromosome depend on its integrity. However, recent experiments suggest that such breakage may occur frequently during "normal" growth in various organisms - from bacteria through vertebrates, p...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-4-5
更新日期:2003-04-28 00:00:00
abstract:BACKGROUND:Core promoters are cis-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-12-32
更新日期:2011-08-01 00:00:00
abstract:BACKGROUND:It is not clear how foreign DNA molecules insert into the host genome. Recently, we have produced transgenic mice to investigate the role of the fad2 gene in the conversion of oleic acid to linoleic acid. Here we describe an integration mechanism of fad2 transgene by homologous illegitimate random integratio...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-11-58
更新日期:2010-08-13 00:00:00
abstract:BACKGROUND:Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping ...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-9-102
更新日期:2008-11-12 00:00:00
abstract:BACKGROUND:Cryptochromes (CRYs) are a class of flavoprotein blue-light signaling receptors found in plants and animals, and they control plant development and the entrainment of circadian rhythms. They also act as integral parts of the central circadian oscillator in humans and other animals. In mammals, the CLOCK-BMAL...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-11-69
更新日期:2010-09-14 00:00:00
abstract:BACKGROUND:Oxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated ...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/s12867-017-0086-z
更新日期:2017-04-05 00:00:00
abstract:BACKGROUND:Selecting stably expressed reference genes is essential for proper reverse transcription quantitative polymerase chain reaction gene expression analysis. However, this choice is not always straightforward. In the case of differentiating human embryonic stem (hES) cells, differentiation itself introduces chan...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-14-21
更新日期:2013-09-12 00:00:00
abstract:BACKGROUND:Small proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of ba...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-13-20
更新日期:2012-06-25 00:00:00
abstract:BACKGROUND:Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-7-42
更新日期:2006-11-13 00:00:00
abstract:BACKGROUND:Krüppel-like factors (KLFs) are a group of master regulators of gene expression conserved from flies to human. However, scant information is available on either the mechanisms or functional impact of the coupling of KLF proteins to chromatin remodeling machines, a deterministic step in transcriptional regula...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-15-10
更新日期:2014-05-25 00:00:00
abstract:BACKGROUND:Wnt signaling is mediated through 1) the beta-catenin dependent canonical pathway and, 2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in ...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-9-11
更新日期:2008-01-23 00:00:00
abstract:BACKGROUND:In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we use...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-8-112
更新日期:2007-12-19 00:00:00
abstract:BACKGROUND:cDNA-RDA is one of the subtractive cloning techniques used to isolate differentially expressed genes between two complex cDNA populations. In the present study we present a modification of the protocol described by Hubank and Schatz. RESULTS:In the post-hybridization mix, the 5'-ends of homoduplexes of inte...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-3-6
更新日期:2002-04-24 00:00:00
abstract:BACKGROUND:Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner. RESULTS:By expressing the human 14-3-3epsilon, as...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-8-27
更新日期:2007-04-12 00:00:00
abstract:BACKGROUND:Traumatic brain injury models are widely studied, especially through gene expression, either to further understand implied biological mechanisms or to assess the efficiency of potential therapies. A large number of biological pathways are affected in brain trauma models, whose elucidation might greatly benef...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-9-62
更新日期:2008-07-08 00:00:00
abstract:BACKGROUND:Single-stranded DNA-binding proteins are essential cellular components required for the protection, metabolism and processing of single-stranded DNA. Human single-stranded DNA-binding protein 1 (hSSB1) is one such protein, with described roles in genome stability maintenance and transcriptional regulation. A...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/s12867-016-0077-5
更新日期:2016-12-09 00:00:00
abstract:BACKGROUND:Core canonical histones are required in the S phase of the cell cycle to pack newly synthetized DNA, therefore the expression of their genes is highly activated during DNA replication. In mammalian cells, this increment is achieved by both enhanced transcription and 3' end processing. In this paper, we descr...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/s12867-018-0110-y
更新日期:2018-07-27 00:00:00
abstract:BACKGROUND:Previous studies suggest that human RAD9 (hRad9), encoding a DNA damage checkpoint molecule, which is frequently amplified in epithelial tumor cells of breast, lung, head and neck cancer, participates in regulation of the tumor suppressor p53-dependent transactivation of pro-survival P21WAF1. This study exam...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-8-37
更新日期:2007-05-21 00:00:00
abstract:BACKGROUND:Cdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease. RESULTS:Insertion of a TEV protease cleavage site into Cdc23 allows i...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-6-13
更新日期:2005-06-07 00:00:00
abstract:BACKGROUND:Invitrogen Gateway technology exploits the integrase/att site-specific recombination system for directional cloning of PCR products and the subsequent subcloning into destination vectors. One or three DNA segments can be cloned using Gateway or MultiSite Gateway respectively. A vast number of single-site Gat...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-7-46
更新日期:2006-12-06 00:00:00
abstract:BACKGROUND:We reported previously that the cardiac troponin I (cTnI) promoter drives cardiac-specific expression of reporter genes in cardiac muscle cells and in transgenic mice, and that disruption of GATA elements inactivates the cTnI promoter in cultured cardiomyocytes. We have now examined the role of cTnI promoter...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-8-78
更新日期:2007-09-17 00:00:00
abstract:BACKGROUND:Although the complete genome sequences of a large number of organisms have been determined, the exact proteomes need to be characterized. More specifically, the extent to which post-translational processes such as proteolysis affect the synthesized proteins has remained unappreciated. We examined this issue ...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-5-6
更新日期:2004-07-01 00:00:00
abstract:BACKGROUND:Alternative splicing is often subjected to complex regulatory control that involves many protein factors and cis-acting RNA sequence elements. One major challenge is to identify all of the protein players and define how they control alternative expression of a particular exon in a combinatorial manner. The M...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-13-35
更新日期:2012-12-10 00:00:00
abstract:BACKGROUND:ATPase/Helicases and nucleases play important roles in homologous recombination repair (HRR). Many of the mechanistic details relating to these enzymes and their function in this fundamental and complicated DNA repair process remain poorly understood in archaea. Here we employed Sulfolobus islandicus, a hype...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/s12867-015-0030-z
更新日期:2015-02-14 00:00:00
abstract:BACKGROUND:Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is a...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-10-99
更新日期:2009-10-30 00:00:00
abstract:BACKGROUND:Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acc...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-8-96
更新日期:2007-10-25 00:00:00
abstract:BACKGROUND:Naturally occurring tRNAs contain numerous modified nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. In model organisms Escherichia coli and Saccharomyces cerevisiae most enzymes involved in this process have been identified. Interes...
journal_title:BMC molecular biology
pub_type: 杂志文章
doi:10.1186/1471-2199-7-23
更新日期:2006-07-18 00:00:00