Transformation of real-time PCR fluorescence data to target gene quantity

Číkoš, Štefan; Koppel, Juraj

doi:10.1016/j.ab.2008.08.031

Cited by 49 publications

(19 citation statements)

References 68 publications

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“…The slope of a standard curve is mathematically correlated to PCR efficiency according to the equation E = 10 −1/slope −1, where E is the PCR efficiency [24]. A 100% efficiency corresponds to a slope value of −3.32.…”

Section: Resultsmentioning

confidence: 99%

Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

Kang

Kim

et al. 2011

PLoS ONE

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Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, −2.1%, and −13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

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Section: Resultsmentioning

confidence: 99%

Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

Kang

Kim

et al. 2011

PLoS ONE

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“…For miRNA detection, we used a TaqMan MicroRNA Assay Kit (Applied Biosystems, USA) and U6 was used as an internal control. Gene expression was determined utilizing TaqMan reagents (Life Technologies, Gaithersburg, MD, USA) with fluorescence signals being normalized to 18s rRNA utilizing the ddCT method [33]. Primers were synthesized by Invitrogen (Shanghai, China) and shown as follow: (Col-1: forward: 5’-TGGCAAGAACGGAGATGAC-3’; reverse: 5’-TCCAAACCACTGAAACCTCTG-3’); (Fibronectin: forward: 5’- GCACATGTCTCGGGAATGGA-3’; reverse: 5’-ACACGTGCAGGAGCAAATGG-3’); (TGF-β1: forward: 5’-ACATTG ACTTCCGCAAGGAC-3’; reverse: 5’-TAGTACACGATGGGCAGTGG-3’); (Smad7: forward: 5’-GTGGCATACTGGGAGGAGAA-3’; reverse: 5’-GATGGAGAAACCAGGGAACA-3’); (miR-21: forward: 5’-GCACCGTCAAGGCTGAGAAC-3; reverse: 5’- CAGCCCATCGACTGGTG-3’) and (U6: forward: 5’-CTCGCTTCGGCAGCACA-3’; reverse: 5’- AACGCTTCACGAATTTGCGT-3’).…”

Section: Methodsmentioning

confidence: 99%

Mir-21 Promotes Cardiac Fibrosis After Myocardial Infarction Via Targeting Smad7

Yuan

Chen

et al. 2017

Cell Physiol Biochem

191

140

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Background/Aims: Cardiac fibrosis after myocardial infarction (MI) has been identified as an important factor in the deterioration of heart function. Previous studies have demonstrated that miR-21 plays an important role in various pathophysiological processes in the heart. However, the role of miR-21 in fibrosis regulation after MI remains unclear. Methods: To induce cardiac infarction, the left anterior descending coronary artery was permanently ligated of mice. First, we explored the expression of miR-21 in the infarcted zone in mice model of MI via RT-qPCR. Next, we examined the effects of TGF-β1 on miR-21 expression in cardiac fibroblasts (CFs). Then, CFs were infected with miR-21 mimics or miR-21 inhibitors to investigate the effects of miR-21 on the process of CFs activation in vitro. Further, bioinformatics analysis and luciferase reporter assay were performed to identify and validate the target gene of miR-21. At last, in-vivo study was done to confirm MiR-21 regulated myocardial fibrosis after MI in mice. Results: MiR-21 was up-regulated in the infarcted zone after MI in vivo. TGF-β1 treatment increased miR-21 expression in CFs. Overexpression of miR-21 promoted the effects of TGF-β1-induced activation of CFs, evidenced by increased expression of Col-1, α-SMA and F-actin, whereas inhibition of miR-21 attenuated the process of fibrosis. Bioinformatics, Western blot analysis and luciferase reporter assay demonstrated that Smad7 is a direct target of miR-21. In addition, in-vivo study revealed that MiR-21 regulated myocardial fibrosis after MI in mice. Conclusion: These findings suggested that miR-21 has a critical role in CF activation and cardiac fibrosis after MI through via TGF-β/Smad7 signaling pathway. Thus, miR-21 promises to be a potential therapy in treatment of cardiac fibrosis after MI.

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“…An approach that has been widely used is the analysis of differential gene expression in the affected tissue [1]–[4]. Quantitative real-time PCR (RT-qPCR) is currently the gold standard for the quantification of steady-state mRNA levels due to its accuracy and sensitivity [5]–[7]. However, in this type of analysis, an appropriate normalization strategy is required for the correction of experimental variations introduced by pipetting errors, inhibitory compounds, reverse transcription efficiency or quality of starting material [8].…”

Section: Introductionmentioning

confidence: 99%

Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

et al. 2013

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It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

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Transformation of real-time PCR fluorescence data to target gene quantity

Cited by 49 publications

References 68 publications

Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

Mir-21 Promotes Cardiac Fibrosis After Myocardial Infarction Via Targeting Smad7

Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

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