Evaluation of qPCR curve analysis methods for reliable biomarker discovery: Bias, resolution, precision, and implications

Ruijter, Jan M.; Pfaffl, Michael W.; Zhao, Sheng; Spiess, Andrej‐Nikolai; Boggy, Gregory; Blom, Jochen; Rutledge, Robert G.; Sisti, Davide; Lievens, Antoon; Preter, Katleen De; Derveaux, Stefaan; Hellemans, Jan; Vandesompele, Jo

doi:10.1016/j.ymeth.2012.08.011

Cited by 207 publications

(274 citation statements)

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“…The reaction conditions for qPCR were as follows: 95˚C for 2 min, followed by 40 cycles at 95˚C for 20 sec, 60˚C for 30 sec and 72˚C for 30 sec, and a final extension step at 72˚C for 10 min. The relative mRNA abundance was determined using the standard curve method (26,27). The data were analyzed using the 2 -ΔΔCT method (26,27).…”

Section: Detection Of Apoptotic Hepatocytes In Liver Tissuesmentioning

confidence: 99%

“…The relative mRNA abundance was determined using the standard curve method (26,27). The data were analyzed using the 2 -ΔΔCT method (26,27). The primers used for RT-qPCR were as follows: FGL1 (144 bp) forward, 5'-GGTGGGCTAGTCACCAAACA-3' and reverse, One-way analysis of variance was employed for statistical comparisons amongst all groups.…”

Section: Detection Of Apoptotic Hepatocytes In Liver Tissuesmentioning

confidence: 99%

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Bone marrow-derived mesenchymal stem cells attenuate acute liver injury and regulate the expression of fibrinogen-like-protein 1 and signal transducer and activator of transcription 3

Zou

Cai

Chen

et al. 2015

Molecular Medicine Reports

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Abstract. In recent years, bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to exert extensive therapeutic effects on acute liver injury; however, the underlying mechanisms of these effects have remained to be elucidated. The present study focused on the potential anti-apoptotic and pro-regenerative effects of BMSCs in D-galactosamine (D-Gal) and lipopolysaccharide (LPS)-induced acute liver injury in rats. An experimental rat acute liver injury model was established by intraperitoneal injection of D-Gal (400 mg/kg) and LPS (80 µg/kg). BMSCs and an identical volume of saline were administered via the caudal vein 2 h after the D-Gal and LPS challenge. Subsequently, the serum samples were collected to detect the levels of alanine aminotransferase and aspartate aminotransferase. Hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated nick-end labeling assay and immunohistochemical staining were performed to determine apoptosis, regeneration and histological changes of liver sections. Western blotting and reverse transcription-quantitative polymerase chain reaction were performed to detect the protein and mRNA expression levels of fibrinogen-like-protein 1 (FGL1), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), STAT3 and B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) in liver tissue samples. The results indicated that intravenous transplantation of BMSCs significantly decreased the levels of alanine aminotransferase and aspartate aminotransferase, and reduced hepatocellular necrosis and inflammatory cell infiltration. Additionally, a terminal deoxynucleotidyl transferase-mediated nick-end labeling assay and immunohistochemical staining revealed that BMSC treatment reduced hepatocyte apoptosis and enhanced liver regeneration. Furthermore, Bcl-2 expression was increased, whilst the protein expression of Bax was reduced. The expression of FGL1 and p-STAT3 were elevated concurrently with the improvement of liver function. These results demonstrated that BMSCs may provide a promising potential agent for the prevention of acute liver injury via inhibition of hepatocyte apoptosis and acceleration of liver regeneration. The mechanism may be, a least in part, a consequence of the upregulation of FGL1 expression and the induction of STAT3 phosphorylation.

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Section: Detection Of Apoptotic Hepatocytes In Liver Tissuesmentioning

confidence: 99%

Section: Detection Of Apoptotic Hepatocytes In Liver Tissuesmentioning

confidence: 99%

Bone marrow-derived mesenchymal stem cells attenuate acute liver injury and regulate the expression of fibrinogen-like-protein 1 and signal transducer and activator of transcription 3

Zou

Cai

Chen

et al. 2015

Molecular Medicine Reports

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“…Cq is localized on the amplification trajectory in regions where the signal significantly deviates from the background noise. Several Cq methods (13 ), such as the cycle threshold method (Ct method), the second derivative maximum (SDM) method (14 ), and the Cy0 method (15 ), have been introduced. The estimation of Cq values from raw data with high noise is challenging, because the Cq depends on the slope of the amplification curve.…”

confidence: 99%

“…Selected Cq methods such as Cy0 perform a correction on the basis of an estimated AE (14 -19 ). It is accepted that most of the existing models and Cq algorithms affect the Cq substantially (13,20 ).…”

confidence: 99%

“…We investigated 6 qPCR parameters: (a) Cq value derived from the SDM of a 5-parameter sigmoid model (Cq SDM ) (13,30,33 ) (33 ); (e) efficiency of the slidingwindow method (sliwin) (17 ) (implemented in the sliwin function); and (f ) the takeoff point (TOP) where the fluorescence rises significantly above background (14 ) (implemented in the takeoff function).…”

Section: Qpcr Parameters Under Investigationmentioning

confidence: 99%

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Impact of Smoothing on Parameter Estimation in Quantitative DNA Amplification Experiments

et al. 2015

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BACKGROUND: Quantification cycle (Cq) and amplification efficiency (AE) are parameters mathematically extracted from raw data to characterize quantitative PCR (qPCR) reactions and quantify the copy number in a sample. Little attention has been paid to the effects of preprocessing and the use of smoothing or filtering approaches to compensate for noisy data. Existing algorithms largely are taken for granted, and it is unclear which of the various methods is most informative. We investigated the effect of smoothing and filtering algorithms on amplification curve data.

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Regulation of Neisseria meningitidis cytochrome bc₁ components by NrrF, a Fur‐controlled small noncoding RNA

et al. 2017

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NrrF is a small regulatory RNA of the human pathogen Neisseria meningitidis . NrrF was previously shown to repress succinate dehydrogenase ( sdh CDAB ) under control of the ferric uptake regulator (Fur). Here, we provide evidence that cytochrome bc 1 , encoded by the polycistronic mRNA pet ABC , is a NrrF target as well. We demonstrated differential expression of cytochrome bc 1 comparing wild‐type meningococci and meningococci expressing NrrF when sufficient iron is available. Using a gfp ‐reporter system monitoring translational control and target recognition of sRNA in Escherichia coli , we show that interaction between NrrF and the 5′ untranslated region of the pet ABC mRNA results in its repression. The NrrF region essential for repression of pet ABC was identified by site‐directed mutagenesis and is fully conserved among meningococci. Our results provide further insights into the mechanism by which Fur controls essential components of the N. meningitidis respiratory chain. Adaptation of cytochrome bc 1 complex component levels upon iron limitation is post‐transcriptionally regulated via the small regulatory RNA NrrF.

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Evaluation of qPCR curve analysis methods for reliable biomarker discovery: Bias, resolution, precision, and implications

Cited by 207 publications

References 46 publications

Bone marrow-derived mesenchymal stem cells attenuate acute liver injury and regulate the expression of fibrinogen-like-protein 1 and signal transducer and activator of transcription 3

Bone marrow-derived mesenchymal stem cells attenuate acute liver injury and regulate the expression of fibrinogen-like-protein 1 and signal transducer and activator of transcription 3

Impact of Smoothing on Parameter Estimation in Quantitative DNA Amplification Experiments

Regulation of Neisseria meningitidis cytochrome bc₁ components by NrrF, a Fur‐controlled small noncoding RNA

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Evaluation of qPCR curve analysis methods for reliable biomarker discovery: Bias, resolution, precision, and implications

Cited by 207 publications

References 46 publications

Bone marrow-derived mesenchymal stem cells attenuate acute liver injury and regulate the expression of fibrinogen-like-protein 1 and signal transducer and activator of transcription 3

Bone marrow-derived mesenchymal stem cells attenuate acute liver injury and regulate the expression of fibrinogen-like-protein 1 and signal transducer and activator of transcription 3

Impact of Smoothing on Parameter Estimation in Quantitative DNA Amplification Experiments

Regulation of Neisseria meningitidis cytochrome bc1 components by NrrF, a Fur‐controlled small noncoding RNA

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Regulation of Neisseria meningitidis cytochrome bc₁ components by NrrF, a Fur‐controlled small noncoding RNA