Impact of Smoothing on Parameter Estimation in Quantitative DNA Amplification Experiments

Spiess, Andrej‐Nikolai; Deutschmann, Claudia; Burdukiewicz, Michał; Himmelreich, R.; Klat, Katharina; Schierack, Peter; Rödiger, Stefan

doi:10.1373/clinchem.2014.230656

Cited by 31 publications

(25 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…F 1…10 ) and then subtract this value from all fluorescence values prior to parameter estimation. Consequently, most of the published quantification algorithms conduct this data transformation18, along with other pre-processing steps such as smoothing or filtering19.…”

mentioning

confidence: 99%

System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation

Spiess

Rödiger

Burdukiewicz

et al. 2016

Sci Rep

Self Cite

View full text Add to dashboard Cite

Real-time quantitative polymerase chain reaction (qPCR) data are found to display periodic patterns in the fluorescence intensity as a function of sample number for fixed cycle number. This behavior is seen for technical replicate datasets recorded on several different commercial instruments; it occurs in the baseline region and typically increases with increasing cycle number in the growth and plateau regions. Autocorrelation analysis reveals periodicities of 12 for 96-well systems and 24 for a 384-well system, indicating a correlation with block architecture. Passive dye experiments show that the effect may be from optical detector bias. Importantly, the signal periodicity manifests as periodicity in quantification cycle (Cq) values when these are estimated by the widely applied fixed threshold approach, but not when scale-insensitive markers like first- and second-derivative maxima are used. Accordingly, any scale variability in the growth curves will lead to bias in constant-threshold-based Cqs, making it mandatory that workers should either use scale-insensitive Cqs or normalize their growth curves to constant amplitude before applying the constant threshold method.

show abstract

mentioning

confidence: 99%

System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation

Spiess

Rödiger

Burdukiewicz

et al. 2016

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…And it based on a 2-fold dilution series of cDNA (1 : 5, 1 : 10, 1 : 10, 1 : 10, 1 : 20, and 1 : 40). The corresponding qPCR efficiencies (E) were calculated refer to the formula E = 10 -1/slope -1 (Pfaffl 2001;Tellinghuisen 2014;Spiess et al 2015Spiess et al , 2016. Each sample was prepared as two biological replicates, and each reaction was analysed with three technical replications.…”

Section: Methodsmentioning

confidence: 99%

Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua

Liu¹,

Li²,

Lu³

et al. 2019

Plant Prot. Sci.

View full text Add to dashboard Cite

Citation: Liu J., Li P., Lu L., Xie L., Chen X., Zhang B. (2019): Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua. Plant Protect. Sci., 55: 61-71.Abstract: Eight commonly used candidate reference genes, 18S ribosomal RNA (rRNA) (18S), 28S rRNA (28S), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1α), ribosomal protein L7 (RPL7), Alpha-tubulin (α-TUB), and TATA box binding protein-associated factor (TBP), were evaluated under various experimental conditions to assess their suitability in different developmental stages, tissues and herbicide treatments in Avena fatua. The results indicated the most suitable reference genes for the different experimental conditions. For developmental stages, 28S and EF1α were the optimal reference genes, both EF1α and 28S were suitable for experiments of different tissues, whereas for herbicide treatments, GAPDH and ACT were suitable for normalizations of expression data. In addition, GAPDH and EF1α were the suitable reference genes.

show abstract

“…Since mRNA quality is crucial [16], we decided to check obtained cDNA by applying qPCR on representative target genes ADRB1, cTnT, GAPDH, SERCA2, SCN5A, MLC-2v and MLC-2a (Table 2). A qPCR reaction mixture included 250 nM of each dNTP, 0.25 M primer (sequences see Table 3), 20-50 ng template cDNA, 1 × PCR buffer, 2.5 mM • C. Raw data were exported as comma-separated values and processed using RKWard (v. 0.6.5) [17] as described [18][19][20][21]. For melting curve analysis dedicated functionality from the MBmca package [22] (v. 0.0.3-4) was used.…”

Section: Rna Isolation and Cdna Synthesismentioning

confidence: 99%

Development of multiplex PCR systems for expression profiling of human cardiomyocytes induced to proliferate by lentivirus transduction of upcyte genes

George

Rödiger

Schröder

et al. 2016

JCB

View full text Add to dashboard Cite

Abstract.Severe heart diseases such as myocarditis and cardiomyopathy are often characterized by progressive damages of contractile heart tissue which ultimately can lead to terminal heart failure. There is a need for relevant in vitro cultures of human cardiomyocytes to study pathogenic processes and to perform pharmacological testing of new heart drugs. By using the upcyte/EPCC (enhanced primary cell culture) approach for direct multiplication of organ-specific cells, we established proliferating human cardiomyocyte cultures derived from atrial appendages. For qualitative cardiac expression profiling we established a comprehensive set of multiplex PCR assays, selected from a panel of 32 genes, to rapidly screen changes at the transcriptional level in human ventricular and atrial cardiomyocytes. Our multiplex PCR approach revealed some donor variability of native atrial heart tissue that need to be confirmed by further studies with more samples. Our initial studies further indicated that characteristic heart muscle cell markers such as MLC-2a, MLC-2v, CHRM2, ADRB1, DES, EDRNB, Cx40 and KCNA5 were down-regulated when isolated cardiomyocytes were taken into primary cell culture. Compared to native heart tissue, proliferating atrial cardiomyocytes lacked expression of those cardiac markers but still expressed MYCD, GATA-4, Cx43, SERCA2, BNP, Tbx5, EDNRA and ACTB. Surprisingly, atrium-derived cardiomyocytes started to express NFAc4 in passage three, and cardiomyocyte marker expressions of Cx43 and BNP were even increased over cultivation time.In conclusion, our novel multiplex PCR assays should be useful for expression profiling of native heart tissues from patients with different disease conditions and for characterization of in vitro cardiomyocyte cultures.Keywords: Atrial appendages, ACTA1, ACTB ACTC1, ADRB1, ADRB2, ATP2A2, atrial fibrillation, BMP2, CHRM2, cluster analysis, DES, EDNRA, EDNRB, enhanced primary cell cultures (EPCC), GAPDH, GATA4, gene expression, GJA1, GJA5, HPRT1, KCNA5, KCNH2, multiplex PCR, MYL2, MYL7, MYOCD, NFATC4, NKX2-5, NPPA, NPPA, PLN, qPCR, reference genes, RPLP0, SCN5A, TBX5, TNNI3, TNNT2, upcyte genes, VIM Abbreviations EPCC enhanced primary cell cultures HE hematoxylin -eosin qPCR quantitative PCR 1 These authors contributed equally.

show abstract

Impact of Smoothing on Parameter Estimation in Quantitative DNA Amplification Experiments

Cited by 31 publications

References 37 publications

System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation

System-specific periodicity in quantitative real-time polymerase chain reaction data questions threshold-based quantitation

Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua

Development of multiplex PCR systems for expression profiling of human cardiomyocytes induced to proliferate by lentivirus transduction of upcyte genes

Contact Info

Product

Resources

About