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Nitsche A: Guideline to reference gene selection for quantitative realtime PCR
- Biochem Biophys Res Commun
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This is an Open Access article distributed under the terms of the Creative Commons Attribution License
Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays
, 2002
"... We compared the accuracy of microarray measurements obtained with oligonucleotide arrays (GeneChip, Affymetrix) with a laboratory-developed cDNA array by assaying test RNA samples from an experiment using a paradigm known to regulate many genes measured on both arrays. We selected 47 genes represent ..."
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Cited by 101 (3 self)
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We compared the accuracy of microarray measurements obtained with oligonucleotide arrays (GeneChip, Affymetrix) with a laboratory-developed cDNA array by assaying test RNA samples from an experiment using a paradigm known to regulate many genes measured on both arrays. We selected 47 genes represented on both arrays, including both known regulated and unregulated transcripts, and established reference relative expression measurements for these genes in the test RNA samples using quantitative reverse transcriptase realtime PCR (QRTPCR) assays. The validity of the reproducible (average coefficient of variation = 11.8%) QRTPCR measurements were established through application of a new mathematical model. The performance of both array platforms in identifying regulated and non-regulated genes was identical. With either platform, 16 of 17 definitely regulated genes were correctly identified, and no definitely unregulated transcript was falsely identified as regulated. Accuracy of the fold-change measurements obtained with each platform was assessed by determining measurement bias. Both platforms consistently underestimate the relative changes in mRNA expression between experimental and control samples. The bias observed with cDNA arrays was predictable for fold-changes <250-fold by QRTPCR and could be corrected by the calibration function F c = F a(cDNA) ,whereF a(cDNA) is the microarray-determined foldchange comparing experimental with control samples, q is the correction factor and F c is the calibrated value. The bias observed with the commercial oligonucleotide arrays was less predictable and calibration was unfeasible. Following calibration, fold-change measurements generated by custom cDNA arrays were more accurate than those obtained by commercial oligonucleotide ar...
DA: A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 2002
"... Real-time reverse transcription (RT) PCR is currently the most sensitive method for the detection of low-abundance mRNAs. Two relative quantitative methods have been adopted: the standard curve method and the comparative CT method. The latter is used when the amplification efficiency of a reference ..."
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Cited by 101 (0 self)
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Real-time reverse transcription (RT) PCR is currently the most sensitive method for the detection of low-abundance mRNAs. Two relative quantitative methods have been adopted: the standard curve method and the comparative CT method. The latter is used when the amplification efficiency of a reference gene is equal to that of the target gene; otherwise the standard curve method is applied. Based on the simulation of kinetic process of real-time PCR, we have developed a new method for quantitation and normalization of gene transcripts. In our method, the amplification efficiency for each individual reaction is calculated from the kinetic curve, and the initial amount of gene transcript is derived and normalized. Simulation demonstrated that our method is more accurate than the comparative CT method and would save more time than the relative standard curve method. We have used the new method to quantify gene expression levels of nine two-pore potassium channels. The relative levels of gene expression revealed by our quantitative method were broadly consistent with those estimated by routine RT-PCR, but the results also showed that amplification efficiencies varied from gene to gene and from sample to sample. Our method provides a simple and accurate approach to quantifying gene expression level with the advantages that neither construction of standard curve nor validation experiments are needed. © 2002 Elsevier Science (USA) The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of specific mRNAs (1, 2). In contrast with classic techniques, such as Northern blot analysis and RNase protection assays, which require large amounts of total
Dobbie Z: Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques 2002, 32(6):1372-1374, 1376, 1378-1379. doi:10.1186/1471-2121-11-68 Cite this article as: Beese et al.: Effect of cAMP derivates on assembly and mainten
- Inclusion in PubMed, CAS, Scopus and Google Scholar • Research
"... Real-time quantitative PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of ..."
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Cited by 97 (0 self)
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Real-time quantitative PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by real-time quantitative PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft ® Excel ®-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of real-time quantitative PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex realtime quantitative PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.
Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl Environ Microbiol 71
, 2005
"... Here we describe a quantitative PCR-based approach to estimating the relative abundances of major taxonomic groups of bacteria and fungi in soil. Primers were thoroughly tested for specificity, and the method was applied to three distinct soils. The technique provides a rapid and robust index of mic ..."
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Cited by 94 (6 self)
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Here we describe a quantitative PCR-based approach to estimating the relative abundances of major taxonomic groups of bacteria and fungi in soil. Primers were thoroughly tested for specificity, and the method was applied to three distinct soils. The technique provides a rapid and robust index of microbial community structure. The diversity of the bacterial and fungal communities in soil is extraordinary (8, 27, 29). High levels of bacterial and fungal diversity make quantifying and characterizing soil microbial communities a daunting task. In recent years, quantitative PCR (qPCR, also referred to as real-time PCR) has emerged as a promising tool for studying soil microbial communities (10, 12, 20, 28). qPCR is based on the real-time detection of a reporter molecule whose fluorescence increases as PCR prod-uct accumulates during each amplification cycle (23). The qPCR approach is somewhat unique among methods of com-munity analysis in that it allows for a relatively rapid yet quan-titative assessment of the abundances of specific phylogenetic groups of microorganisms in soil. In this paper, we describe a qPCR-based approach to as-sessing soil microbial community structure at broad taxonomic levels. We developed and tested nine individual qPCR assays to quantify the abundances of the dominant groups of bacteria and fungi found in the soil environment. qPCR assays were conducted in polypropylene 96-well plates on an ABI Prism 7000 sequence detection system (Ap-plied Biosystems). Each 25-l reaction contained the follow-ing: 12.5 l of ABsolute qPCR Master Mix (ABgene), 1.25 l of each primer (10 M; Invitrogen), 2.5 l bovine serum al-bumin (10 mg ml1; Promega), 1.0 l SYBRGreen dye (1,600-
Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data
- Nucleic Acids Res
, 2009
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1 DISTINCT RIG-I AND MDA5 SIGNALING BY RNA VIRUSES IN INNATE IMMUNITY
"... differentiation associated gene-5; IPS-1, Interferon promoter stimulating factor-1; IRF-3, interferon regulatory factor-3; IFN, interferon; SenV, Sendai virus; NDV, Newcastle disease virus; RSV, respiratory syncytial virus; DEN2, dengue virus type 2 ..."
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Cited by 40 (1 self)
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differentiation associated gene-5; IPS-1, Interferon promoter stimulating factor-1; IRF-3, interferon regulatory factor-3; IFN, interferon; SenV, Sendai virus; NDV, Newcastle disease virus; RSV, respiratory syncytial virus; DEN2, dengue virus type 2
2007 Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair
- Mol Cell Biol
"... Expansions of CAG repeat tracts in the germ line underlie several neurological diseases. In human patients and mouse models, CAG repeat tracts display an ongoing instability in neurons, which may exacerbate disease symptoms. It is unclear how repeats are destabilized in nondividing cells, but it can ..."
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Cited by 27 (3 self)
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Expansions of CAG repeat tracts in the germ line underlie several neurological diseases. In human patients and mouse models, CAG repeat tracts display an ongoing instability in neurons, which may exacerbate disease symptoms. It is unclear how repeats are destabilized in nondividing cells, but it cannot involve DNA replica-tion. We showed previously that transcription through CAG repeats induces their instability (Y. Lin, V. Dion, and J. H. Wilson, Nat. Struct. Mol. Biol. 13:179–180). Here, we present a genetic analysis of the link between transcription-induced repeat instability and nucleotide excision repair (NER) in human cells. We show that short interfering RNA-mediated knockdown of CSB, a component specifically required for transcription-coupled NER (TC-NER), and knockdowns of ERCC1 and XPG, which incise DNA adjacent to damage, stabilize CAG repeat tracts. These results suggest that TC-NER is involved in the pathway for transcription-induced CAG repeat instability. In contrast, knockdowns of OGG1 and APEX1, key components involved in base excision repair, did not affect repeat instability. In addition, repeats are stabilized by knockdown of transcrip-tion factor IIS, consistent with a requirement for RNA polymerase II (RNAPII) to backtrack from a tran-scription block. Repeats also are stabilized by knockdown of either BRCA1 or BARD1, which together function as an E3 ligase that can ubiquitinate arrested RNAPII. Treatment with the proteasome inhibitor MG132, which stabilizes repeats, confirms proteasome involvement. We integrate these observations into a tentative pathway
Role of RcsF in Signaling to the Rcs Phosphorelay Pathway in Escherichia coli†
, 2005
"... This article cites 43 articles, 33 of which can be accessed free ..."
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Cited by 25 (0 self)
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This article cites 43 articles, 33 of which can be accessed free
DP: SYBR green-based real-time quantitative PCR assay for detection of West Nile Virus circumvents false-negative results due to strain variability
- J Clin Microbiol
"... This article cites 29 articles, 18 of which can be accessed free at: ..."
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Cited by 25 (4 self)
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This article cites 29 articles, 18 of which can be accessed free at:
