ArticleMarine biotechnology (New York, N.Y.)2011
Normalizing RT-qPCR data: are we getting the right answers? An appraisal of normalization approaches and internal reference genes from a case study in the finfish Lates calcarifer.
Article in Marine biotechnology (New York, N.Y.), 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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25 citing papers in PubMed, 78 citations in OpenAlex.
- Identification of Reference Genes for RT-qPCR Assays inCurrent issues in molecular biology · 2026Article
- Changes in gene expression at the early stages of barramundi (Lates calcarifer) muscle cell line development.Molecular biology reports · 2026Article
- Genome assembly of the Neotropical marsh rat Holochilus nanus (Cricetidae: Sigmodontinae) brings insights on B and sex chromosome evolution.Mammalian genome : official journal of the International Mammalian Genome Society · 2026Article
- Freshwater transfer affected intestinal microbiota with correlation to cytokine gene expression in Asian sea bass.Frontiers in microbiology · 2023Article
- First characterization of PIWI-interacting RNA clusters in a cichlid fish with a B chromosome.BMC biology · 2022Article
- Screening of reference genes in tiger puffer (Takifugu rubripes) across tissues and under different nutritional conditions.Fish physiology and biochemistry · 2021Article
- Differential expression of miRNAs in the presence of B chromosome in the cichlid fish Astatotilapia latifasciata.BMC genomics · 2021Article
- Article
- Adequate supply of dietary taurine stimulates expression of molecular markers of growth and protein turnover in juvenile barramundi (Lates calcarifer).Fish physiology and biochemistry · 2020Article
- Sex-specific dmrt1 and cyp19a1 methylation and alternative splicing in gonads of the protandrous hermaphrodite barramundi.PloS one · 2018Article
- Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice.International journal of molecular sciences · 2017Article
- Eicosapentaenoic Acid, Arachidonic Acid and Eicosanoid Metabolism in Juvenile Barramundi Lates calcarifer.Lipids · 2016Article
- Article
- Identification of Novel Reference Genes Suitable for qRT-PCR Normalization with Respect to the Zebrafish Developmental Stage.PloS one · 2016Article
- Identification of Barramundi (Lates calcarifer) DC-SCRIPT, a Specific Molecular Marker for Dendritic Cells in Fish.PloS one · 2015Article
- Persian sturgeon insulin-like growth factor I: molecular cloning and expression during various nutritional conditions.Journal of applied genetics · 2014Article
- Postprandial molecular responses in the liver of the barramundi, Lates calcarifer.Fish physiology and biochemistry · 2014Article
- Comprehensive selection of reference genes for gene expression normalization in sugarcane by real time quantitative rt-PCR.PloS one · 2014Article
- Isolation and selection of suitable reference genes for real-time PCR analyses in the skeletal muscle of the fine flounder in response to nutritional status: assessment and normalization of gene expression of growth-related genes.Fish physiology and biochemistry · 2013Article
- Gelatinases impart susceptibility to high-fat diet-induced obesity in mice.The Journal of nutritional biochemistry · 2013Article
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3 authors at 1 institution in 1 country.
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Abstract
Commonly used normalization approaches for quantitative reverse transcription polymerase chain reaction analyses include (a) input nucleic acids standardization (ΔC (q) method), (b) normalizing target gene transcript abundance against a single internal reference gene (ΔΔC (q) method), and (c) geometric averaging of multiple reference gene abundance using the geNorm software. We compared these three approaches to examine expression of a negative muscle growth regulator gene, myostatin-I (mstn-I), in the finfish Lates calcarifer, following 4 weeks of nutritional fasting. Interestingly, these three different approaches led to widely divergent data interpretations. When ΔC (q) and subsequently ΔΔC (q) with α-tub as the reference gene were applied to mstn-I expression data, an ∼threefold upregulation of this gene was observed in fasted compared to fed fish. In contrast, mstn-I appeared unchanged when data was normalized against the geometric average of the two apparently most "stable" reference genes (elongation factor-1 α (ef1-α) and rpl8) selected from a panel comprising seven commonly utilized candidate reference genes (18S, cat-D, ef1-α, rpl8, gapdh, ubq, and α-tub). The geNorm software erroneously suggested that ef1-α, rpl8, and ubq were the most "stable" reference genes, whereas ΔC (q) analysis revealed these genes simply to exhibit similar patterns of regulation in response to fasting. The ΔC (q) approach showed that α-tub was the least variable in its expression level between fasted and fed fish after 4 weeks. The present study also highlights the importance of validating internal references for each time point under investigation when applying ΔΔC (q) and suggests that the more cost-effective ΔC (q) normalization approach, if carefully applied, may in fact produce the most biologically valid results.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.