ArticlePloS one2012
Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.
Article in PloS one, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 48 papers, 3 of them syntheses 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
48 citing papers in PubMed, 3 syntheses or guidelines pooled it, 83 citations in OpenAlex.
- A systematic review on the selection of reference genes for gene expression studies in rodents: are the classics the best choice?Molecular biology reports · 2024Pooled it
- Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)Autophagy · 2021Guideline
- With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.PloS one · 2015Pooled it
- Molecular effects of digital psychological intervention for perinatal stress: cell culture, animal model validation, and machine learning-based biomarker identification.Scientific reports · 2026Article
- Systems Biology Approach to Unraveling Transcriptomic Mechanisms of Ganfule Capsules in Ameliorating Nonalcoholic Fatty Liver Disease.Combinatorial chemistry & high throughput screening · 2026Article
- USH2A-Mutated Human Retinal Organoids Model Rod-Cone Dystrophy.Investigative ophthalmology & visual science · 2025Article
- Tree Shade Improves Milking Performance, Apparent Digestibility, Antioxidant Capacity, and Immunity of Dairy Cows in Open Sheds.Animals : an open access journal from MDPI · 2025Article
- Genetic mechanisms of hemispheric functional connectivity in diabetic retinopathy: a joint neuroimaging and transcriptomic study.Frontiers in cell and developmental biology · 2025Article
- Conditional Knockouts of Interphotoreceptor Retinoid Binding Protein Suggest Two Independent Mechanisms for Retinal Degeneration and Myopia.Investigative ophthalmology & visual science · 2024Article
- Reference genes to study the sex-biased expression of genes regulating Drosophila metabolism.Scientific reports · 2024Article
- Influence of maternal nutrition and one-carbon metabolites supplementation on bovine antimicrobial peptides in fetal and maternal tissues.Frontiers in veterinary science · 2024Article
- Identification of reference genes for expression studies in the liver and spleen of laying hens housed in cage and cage-free systems.Open veterinary journal · 2023Article
- Article
- Retinal Gene Expression of Selective Genes and Histological Stages of Embryonic and Post-Hatch Chickens (Genes · 2022Article
- Downregulation of NCL attenuates tumor formation and growth in HeLa cells by targeting the PI3K/AKT pathway.Cancer medicine · 2022Article
- microRNA-211-5p predicts the progression of postmenopausal osteoporosis and attenuates osteogenesis by targeting dual specific phosphatase 6.Bioengineered · 2022Article
- Article
- Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea.Scientific reports · 2020Article
- Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries.Journal of assisted reproduction and genetics · 2020Article
- Prostate cancer cells hyper-activate CXCR6 signaling by cleaving CXCL16 to overcome effect of docetaxel.Cancer letters · 2019Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
3 authors at 1 institution in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundHousekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. METHODOLOGY/PRINCIPAL
findingsWe applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed.
conclusionPutative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.
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.