ArticlePloS one2015
Reference gene validation for RT-qPCR, a note on different available software packages.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 140 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
140 citing papers in PubMed, 1 synthesis or guideline pooled it.
- Selection of reference genes for quantitative PCR: identifying reference genes for airway epithelial cells exposed toAmerican journal of physiology. Lung cellular and molecular physiology · 2020Pooled it
- A plasma circulating miRNAs profile predicts type 2 diabetes mellitus and prediabetes: from the CORDIOPREV study.Experimental & molecular medicine · 2018Trial
- Validation of Optimal Reference Genes for qRT-PCR in Adipose- and Uterine-Derived Feline Mesenchymal Stem Cells.Veterinary sciences · 2026Article
- Screening of Stable Reference Genes inVeterinary sciences · 2026Article
- Article
- Identification and validation of reference genes for quantitative real-time PCR in Corydalis saxicola under various abiotic stresses.Scientific 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
- Identification of a novel real-time PCR reference gene panel for EA.hy926 endothelial cells in different growth States and with DHA treatment.Scientific reports · 2026Article
- Validating the classics: Accurate reference gene panel for reliable RT-qPCR in Porifera.PloS one · 2026Article
- Advances in algorithms for normalizer gene selection in qRT-PCR: implications for cancer biology and precision medicine.Frontiers in genetics · 2026Review
- Selection and validation of suitable reference genes for qPCR expression analysis in youngFrontiers in plant science · 2026Article
- Validation of Housekeeping Genes for Normalizing RNA Expression in Real-Time PCR in Tuberculomas and Peripheral Blood Mononuclear Cells for Pulmonary Tuberculosis Patients.International journal of molecular sciences · 2025Article
- Screening and Identification of Reference Genes Under Different Conditions and Growth Stages ofInternational journal of molecular sciences · 2025Article
- Developmental and environmental stability of candidate reference genes in the wild bee ceratina calcarata.Scientific reports · 2025Article
- Stabilizing the Baseline: Reference Gene Evaluation in Three InvasiveInternational journal of molecular sciences · 2025Article
- Selection of Stable Reference Genes for Gene Expression Studies in Activated and Non-Activated PBMCs Under Normoxic and Hypoxic Conditions.International journal of molecular sciences · 2025Article
- Reference genes selection and validation in yam by real-time quantitative polymerase chain reaction.Scientific reports · 2025Article
- Selection and Validation of Reference Genes inInternational journal of molecular sciences · 2025Article
- Identifying the best reference gene for RT-qPCR analyses of the three-dimensional osteogenic differentiation of human induced pluripotent stem cells.Bone reports · 2024Article
- Guidelines for mitochondrial RNA analysis.Molecular therapy. Nucleic acids · 2024Review
80 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
11 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundAn appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms.
results14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells (eBM-MSC). Expression stabilities of the reference genes were evaluated using geNorm, NormFinder, and BestKeeper. Similar results were obtained by the three approaches for the most and least stably expressed genes. The R-based packages NormqPCR, SLqPCR and the NormFinder for R script gave identical gene rankings. Interestingly, different outputs were obtained between the original software packages and the RefFinder tool, which is based on raw Cq values for input. When the raw data were reanalysed assuming 100% efficiency for all genes, then the outputs of the original software packages were similar to the RefFinder software, indicating that RefFinder outputs may be biased because PCR efficiencies are not taken into account.
conclusionsThis report shows that assay efficiency is an important parameter for reference gene validation. New software tools that incorporate these algorithms should be carefully validated prior to use.
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.