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Selecting suitable reference genes for qPCR normalization: a comprehensive analysis in MCF-7 breast cancer cell line
- Source :
- BMC Molecular and Cell Biology, Vol 21, Iss 1, Pp 1-19 (2020), BMC Molecular and Cell Biology
- Publication Year :
- 2020
- Publisher :
- Springer Science and Business Media LLC, 2020.
-
Abstract
- Background MCF-7 breast cancer cell line is undoubtedly amongst the most extensively studied patient-derived research models, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, have previously identified suitable reference genes for qPCR normalization in MCF-7 cell line. However, over the course of identification of suitable reference genes, a comparative analysis comprising these genes together in a single study has not been reported. Furthermore, the expression dynamics of these reference genes within sub-clones cultured over multiple passages (p) has attracted limited attention from research groups. Therefore, we investigated the expression dynamics of 12 previously suggested reference genes within two sub-clones (culture A1 and A2) cultured identically over multiple passages. Additionally, the effect of nutrient stress on reference gene expression was examined to postulate an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies. Results The analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as the least variable reference genes while for culture A2, GAPDH-RNA28S were identified. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization of AURKA and KRT19 with triplet pair GAPDH-CCSER2-PCBP1 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress. Conclusions The variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-CCSER2-PCBP1 triplet offers a reliable alternative to otherwise traditionally used internal controls for optimizing intra- and inter-assay gene expression differences. Furthermore, we suggest avoiding the use of ACTB, GAPDH and PGK1 as single internal controls.
- Subjects :
- Culture media
0301 basic medicine
Normalization (statistics)
Breast Neoplasms
Computational biology
Biology
Real-Time Polymerase Chain Reaction
03 medical and health sciences
0302 clinical medicine
Breast cancer cell line
Cell Line, Tumor
Reference genes
Gene expression
Humans
lcsh:QH573-671
Molecular Biology
Gene
Aurora Kinase A
Sub-clones
Keratin-19
lcsh:Cytology
Gene Expression Profiling
RT-qPCR
Nutrient stress
RNA-Binding Proteins
Cell Biology
Reference Standards
DNA-Binding Proteins
030104 developmental biology
MCF-7
Cell culture
030220 oncology & carcinogenesis
MCF-7 Cells
Female
Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
Research Article
Subjects
Details
- ISSN :
- 26618850
- Volume :
- 21
- Database :
- OpenAIRE
- Journal :
- BMC Molecular and Cell Biology
- Accession number :
- edsair.doi.dedup.....9a884e584d5fc969b9c32047febc6344