Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis

Department of Integrative and Molecular Neuroscience, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College London, Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK.
Nucleic Acids Research (Impact Factor: 9.11). 08/2003; 31(14):e73.
Source: PubMed


Real-time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. Despite a wide range of approaches, the same principles underlie all data analysis, with standard approaches broadly classified as either absolute or relative. In this study we use a variety of absolute and relative approaches of data analysis to investigate nocturnal c-fos expression in wild-type and retinally degenerate mice. In addition, we apply a simple algorithm to calculate the amplification efficiency of every sample from its amplification profile. We confirm that nocturnal c-fos expression in the rodent eye originates from the photoreceptor layer, with around a 5-fold reduction in nocturnal c-fos expression in mice lacking rods and cones. Furthermore, we illustrate that differences in the results obtained from absolute and relative approaches are underpinned by differences in the calculated PCR efficiency. By calculating the amplification efficiency from the samples under analysis, comparable results may be obtained without the need for standard curves. We have automated this method to provide a means of streamlining the real-time PCR process, enabling analysis of experimental samples based upon their own reaction kinetics rather than those of artificial standards.

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    • "Data analysis for real-time PCR (DART-PCR) (Peirson et al., 2003) was used for analysing the raw qPCR data. For every sample, DART- PCR enables calculation of threshold cycles and amplification efficiencies. "
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    ABSTRACT: The molecular mechanisms activated by environmental contaminants and natural stressors such as freezing need to be investigated in order to better understand the mechanisms of interaction and potential effects that combined stressors may have on organisms. Using the freeze tolerant earthworm Dendrobaena octaedra as model species, we exposed worms to freezing and exposure to sublethal copper in a factorial design and investigated the transcription of candidate genes for metal and cold stress. We hypothesized that both freezing and copper would induce transcription of genes coding for heat shock proteins (hsp10 and hsp70), metallothioneins (mt1 and mt2) and glutathione-S-transferase (gst), and that the combined effects of these two stressors would be additive. The gene transcripts hsp10, hsp70 and gst were significantly upregulated by freezing, but only hsp10 was upregulated by copper. We found that copper at the time of sampling had no effect on transcription of two metallothionein genes whereas transcription was strongly upregulated by freezing. Moreover there was a significant interaction causing more than additive transcription rates of mt1 in the copper/freezing treatment suggesting that freeze-induced cellular dehydration increases the concentration of free copper ions in the cytosol. This metallothionein response to freezing is likely adaptive and possibly provides protection against freeze-induced elevated metal concentrations in the cytosol and excess ROS levels due to hypoxia during freezing. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · Aug 2015 · Comparative Biochemistry and Physiology Part C Toxicology & Pharmacology
    • "PCR conditions were as follows: 95 C for 3 min followed by 40 cycles of 95 C for 15 s, 58 C for 30 s, and 95 C for 10 s. For comparing the expression levels of the different genes, R 0 values were determined as described by Peirson et al. (2003). To determine the relative expression levels, the DDC T method was used (Livak and Schmittgen, 2001). "

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    • " doi:10.1371/journal.pone.0125672.t002 [29] . Since the results for the other 11 miRNA candidates were consistent between the microarray and qRT-PCR analysis, the validity of the microarray screen was proved. Our study not only confirmed some of the results from previous studies, but further revealed many novel miRNAs that have not been previously reported in ccRCC tumorigenesis."
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    ABSTRACT: Objective: This study aims to profile dysregulated microRNA (miRNA) expression in clear cell renal cell carcinoma (ccRCC) and to identify key regulatory miRNAs in ccRCC. Methods and results: miRNA expression profiles in nine pairs of ccRCC tumor samples at three different stages and the adjacent, non-tumorous tissues were investigated using miRNA arrays. Eleven miRNAs were identified to be commonly dysregulated, including three up-regulated (miR-487a, miR-491-3p and miR-452) and eight down-regulated (miR-125b, miR-142-3p, miR-199a-5p, miR-22, miR-299-3p, miR-29a, miR-429, and miR-532-5p) in tumor tissues as compared with adjacent normal tissues. The 11 miRNAs and their predicted target genes were analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and three key miRNAs (miR-199a-5p, miR-22 and miR-429) were identified by microRNA-gene network analysis. Dysregulation of the three key miRNAs were further validated in another cohort of 15 ccRCC samples, and the human kidney carcinoma cell line 786-O, as compared with five normal kidney samples. Further investigation showed that over-expression of miR-199a-5p significantly inhibited the invasion ability of 786-O cells. Luciferase reporter assays indicated that miR-199a-5p regulated expression of TGFBR1 and JunB by directly interacting with their 3' untranslated regions. Transfection of miR-199a-5p successfully suppressed expression of TGFBR1 and JunB in the human embryonic kidney 293T cells, further confirming the direct regulation of miR-199a-5p on these two genes. Conclusions: This study identified 11 commonly dysregulated miRNAs in ccRCC, three of which (miR-199a-5p, miR-22 and miR-429) may represent key miRNAs involved in the pathogenesis of ccRCC. Further studies suggested that miR-199a-5p plays an important role in inhibition of cell invasion of ccRCC cells by suppressing expression of TGFBR1 and JunB.
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