ArticleBMC genomics2014
Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset.
Article in BMC genomics, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 50 papers, 1 of them a synthesis that pooled it.
What it found
Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.
The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.
Who cites it
50 citing papers in PubMed, 1 synthesis or guideline pooled it, 136 citations in OpenAlex.
- With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.PloS one · 2015Pooled it
- A crossover randomized controlled trial examining the effects of black seed (Nigella sativa) supplementation on IL-1β, IL-6 and leptin, and insulin parameters in overweight and obese women.BMC complementary medicine and therapies · 2024Trial
- Screening of Stable Reference Genes inVeterinary sciences · 2026Article
- Selection and evaluation of reference genes for quantitative real-time polymerase chain reaction normalization in Pieris melete (Lepidoptera, Pieridae).Journal of insect science (Online) · 2025Article
- Selection and validation of reference genes for RT-qPCR normalization in dormant cancer cells.Scientific reports · 2025Article
- Selection and Validation of Reference Genes inInternational journal of molecular sciences · 2025Article
- Selection and Validation of qRT-PCR Internal Reference Genes to Study Flower Color Formation inInternational journal of molecular sciences · 2024Article
- Seasonal Stability Assessment of Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Normalization inCurrent issues in molecular biology · 2024Article
- Early Molecular Immune Responses of Turbot (International journal of molecular sciences · 2023Article
- YWHAZ and TBP are potential reference gene candidates for qPCR analysis of response to radiation therapy in colorectal cancer.Scientific reports · 2023Article
- Extraction of High-Quality RNA fromMicroorganisms · 2023Article
- Improvement of crAssphage detection/quantification method and its extensive application for food safety.Frontiers in microbiology · 2023Article
- Selection of species specific panel of reference genes in peripheral blood mononuclear cells of native livestock species adapted to trans-Himalayan region of Leh-Ladakh.Scientific reports · 2022Article
- Article
- Identification and evaluation of an appropriate housekeeping gene for real time gene profiling of hepatocellular carcinoma cells cultured in three dimensional scaffold.Molecular biology reports · 2022Article
- Comparative Transcriptome Sequencing Analysis ofFrontiers in physiology · 2022Article
- Screening of reference genes in tiger puffer (Takifugu rubripes) across tissues and under different nutritional conditions.Fish physiology and biochemistry · 2021Article
- Research on the Biofilm Formation ofMicroorganisms · 2021Article
- Selection and Validation of Reference Genes for Quantitative RT-PCR Analysis inPlants (Basel, Switzerland) · 2021Article
- Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea.Scientific reports · 2020Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
7 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundGene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry.
resultsWe analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency.
conclusionOur results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively. We also recommend the use of UBQ and RPS4 for normalization of gonad development samples in turbot.
Indexed as
Identifiers
What OpenQuestion holds
Registered trials
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.