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Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos
Published on: February 22, 2016
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Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo
John C W Hildyard1, Dominic J Wells2, Richard J Piercy1
1Comparative Neuromuscular Diseases Laboratory, Clinical Science and Services, Royal Veterinary College, London, NW1 0TU, UK.
Wellcome Open Research
|September 29, 2022
Summary
Identifying stable reference genes for mouse embryonic gene expression studies is crucial. AP3D1, RPL13A, and PAK1IP1 are recommended as universally suitable reference genes for quantitative polymerase chain reaction (qPCR) analysis in developing mouse embryos.
Area of Science:
- Developmental Biology
- Genomics
- Molecular Biology
Background:
- Mammalian embryogenesis involves complex cellular interactions and lineage differentiation.
- Quantitative polymerase chain reaction (qPCR) is vital for studying gene expression during development.
- Accurate qPCR requires normalization using stably expressed reference genes, which are challenging to identify in dynamic embryonic systems.
Purpose of the Study:
- To identify reliable reference genes for gene expression analysis in mouse embryos during mid to late gestation (E11.5-E18.5).
- To evaluate the expression stability of 15 candidate reference genes across whole embryos and specific tissues (head, forelimb).
- To determine the most suitable reference genes for accurate normalization in developmental studies.
Main Methods:
- Expression stability analysis of 15 candidate reference genes in mouse embryos (E11.5-E18.5).
- Utilized whole embryo, head, and forelimb samples for gene expression profiling.
- Employed four algorithms (geNorm, Normfinder, Bestkeeper, deltaCt) to assess gene stability.
- Evaluated genes including ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M, and PAK1IP1.
Main Results:
- Multiple candidate genes demonstrated acceptable expression stability across different embryonic samples.
- AP3D1, RPL13A, and PAK1IP1 emerged as the top-performing reference genes, showing high stability overall.
- HPRT1 and B2M exhibited significant developmental regulation, making them unsuitable as reference genes.
- Normalization with top-ranked references revealed subtle expression patterns in genes like CDC40 and HTATSF1.
Conclusions:
- AP3D1, RPL13A, and PAK1IP1 are recommended as universally suitable reference genes for gene expression studies in mouse embryos from E11.5 to E18.5.
- These genes provide reliable normalization for accurate analysis of developmental gene expression.
- The findings enhance the reliability of qPCR-based research in mammalian embryogenesis.
