ArticlePloS one2012
Pseudogenes as weaknesses of ACTB (Actb) and GAPDH (Gapdh) used as reference genes in reverse transcription and polymerase chain reactions.
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 64 papers, 1 of them a synthesis that pooled it.
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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.
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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.
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Who cites it
64 citing papers in PubMed, 1 synthesis or guideline pooled it, 104 citations in OpenAlex.
- With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.PloS one · 2015Pooled it
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- Engineered iMSCs delivering wild-type IL-2 achieve dose-sparing tumor control via CD25-dependent CD8BMC medicine · 2026Article
- The good, the bad, and the stable: Reference genes for preclinical biodistribution studies.Molecular therapy. Advances · 2026Article
- Matrix- and Differentiation Stage-Dependent Variability of Reference Genes: Rethinking Validation Strategies in 3T3-L1 Adipogenic Models.International journal of molecular sciences · 2026Article
- Validating the classics: Accurate reference gene panel for reliable RT-qPCR in Porifera.PloS one · 2026Article
- Selecting Optimal Housekeeping Genes for RT-qPCR in Endometrial Cancer Studies: A Narrative Review.International journal of molecular sciences · 2025Review
- Development, optimization and application of a universal fluorescence multiplex PCR-based assay to detect BCOR genetic alterations in pediatric tumors.Diagnostic pathology · 2025Article
- Expression patterns of housekeeping genes and tissue-specific genes in black goats across multiple tissues.Scientific reports · 2024Article
- Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations.iScience · 2024Article
- Diet xylo-oligosaccharide supplementation improves growth performance, immune function, and intestinal health of broilers.Animal nutrition (Zhongguo xu mu shou yi xue hui) · 2024Article
- Analysis of the impact of DGAT1 p.M435L and p.K232A variants on pre-mRNA splicing in a full-length gene assay.Scientific reports · 2023Article
- Residual Humidity in Paraffin-Embedded Tissue Reduces Nucleic Acid Stability.International journal of molecular sciences · 2023Article
- Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans.International journal of molecular sciences · 2023Article
- A Strategy for the Selection of RT-qPCR Reference Genes Based on Publicly Available Transcriptomic Datasets.Biomedicines · 2023Article
- Effects of Dermatan Sulfate from Marine InvertebrateMarine drugs · 2022Article
- Expression and Prognostic Value of Melanoma-Associated Antigen D2 in Gliomas.Brain sciences · 2022Article
- Multiplex SuperSelective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations in Liquid Biopsies.The Journal of molecular diagnostics : JMD · 2022Article
- New insights into the role of ribonuclease P protein subunit p30 from tumor to internal reference.Frontiers in oncology · 2022Review
- Article
4 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
4 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The genes encoding β-actin (ACTB in human or Actb in mouse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH in human or Gapdh in mouse) are the two most commonly used references for sample normalization in determination of the mRNA level of interested genes by reverse transcription (RT) and ensuing polymerase chain reactions (PCR). In this study, bioinformatic analyses revealed that the ACTB, Actb, GAPDH and Gapdh had 64, 69, 67 and 197 pseudogenes (PGs), respectively, in the corresponding genome. Most of these PGs are intronless and similar in size to the authentic mRNA. Alignment of several PGs of these genes with the corresponding mRNA reveals that they are highly homologous. In contrast, the hypoxanthine phosphoribosyltransferase-1 gene (HPRT1 in human or Hprt in mouse) only had 3 or 1 PG, respectively, and the mRNA has unique regions for primer design. PCR with cDNA or genomic DNA (gDNA) as templates revealed that our HPRT1, Hprt and GAPDH primers were specific, whereas our ACTB and Actb primers were not specific enough both vertically (within the cDNA) and horizontally (compared cDNA with gDNA). No primers could be designed for the Gapdh that would not mis-prime PGs. Since most of the genome is transcribed, we suggest to peers to forgo ACTB (Actb) and GAPDH (Dapdh) as references in RT-PCR and, if there is no surrogate, to use our primers with extra caution. We also propose a standard operation procedure in which design of primers for RT-PCR starts from avoiding mis-priming PGs and all primers need be tested for specificity with both cDNA and gDNA.
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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.