Identification of suitable endogenous control genes for microRNA expression profiling of childhood medulloblastoma and human neural stem cells
Open accessAbstract
Background: Medulloblastoma (MB) is the most common type of malignant childhood brain tumour. Although deregulated microRNA (miRNA) expression has been linked to MB pathogenesis, the selection of appropriate candidate endogenous control (EC) reference genes for MB miRNA expression profiling studies has not been systematically addressed. In this study we utilised reverse transcriptase quantitative PCR (RT-qPCR) to identify the most appropriate EC reference genes for the accurate normalisation of miRNA expression data in primary human MB specimens and neural stem cells. Results: Expression profiling of 662 miRNAs and six small nuclear/ nucleolar RNAs in primary human MB specimens, two CD133+ neural stem cell (NSC) populations and two CD133- neural progenitor cell (NPC) populations was performed using TaqMan low-density array (TLDA) cards. Minimal intra-card variability for candidate EC reference gene replicates was observed, however significant inter-card variability was identified between replicates present on both TLDA cards A and B. A panel of 18 potentially suitable EC reference genes was identified for the normalisation of miRNA expression on TLDA cards. These candidates were not significantly differentially expressed between CD133+ NSCs/ CD133- NPCs and primary MB specimens. Of the six sn/snoRNA EC reference genes recommended by the manufacturer, only RNU44 was uniformly expressed between primary MB specimens and CD133+ NSC/CD133- NPC populations ( P = 0.709; FC = 1.02). The suitability of candidate EC reference genes was assessed using geNorm and NormFinder software, with hsa-miR-301a and hsa-miR-339-5p found to be the most uniformly expressed EC reference genes on TLDA card A and hsa-miR-425* and RNU24 for TLDA card B. Conclusions: A panel of 18 potential EC reference genes that were not significantly differentially expressed between CD133+ NSCs/ CD133- NPCs and primary human MB specimens was identified. The top ranked EC reference genes described here should be validated in a larger cohort of specimens to verify their utility as controls for the normalisation of RT-qPCR data generated in MB miRNA expression studies. Importantly, inter-card variability observed between replicates of certain candidate EC reference genes has major implications for the accurate normalisation of miRNA expression data obtained using the miRNA TLDA platform.
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All reactions for the study were pipetted into a single 96-well plate each for ddPCR and qPCR to eliminate inter-plate variability.
“Therefore, all reactions for the study were pipetted into a single 96-well plate each for ddPCR and qPCR technologies.”
From Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data (2017) · cites this paper as [16]
The Results section on inter-card variation reports significant differences between some replicates on separate TLDA cards and minimal variation within a card, supporting the rationale for limiting inter-plate variability (page unavailable). The Methods section describes RT-qPCR on TLDA cards, not ddPCR or reactions pipetted into single 96-well plates; the paper does not establish that the citing study used those plates. (LLM, conf=0.86)
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Corrêa, C. A. P., Chagas, P. S., Baroni, M., Andrade, A. F., Queiróz, R. G. D. P., Suazo, V. K., Cruzeiro, G. A. V., Fedatto, P. F., Antônio, D. S. M., Brandalise, S. R., et al. (2025) · The Cerebellum · doi:10.1007/s12311-025-01812-3 · Cited by 3 · see citing works
Ching, A.-S., & Ahmad‐Annuar, A. (2015) · Sains Malaysiana · doi:10.17576/jsm-2015-4410-14 · Cited by 3 · see citing works
Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models.
Qiao, G., Li, Q., Peng, G., Ma, J., Fan, H., & Li, Y. (2013) · PubMed Central · doi:10.3969/j.issn.1673-5374.2013.25.007
- Authors
- Genovesi, L. A., Anderson, D., Carter, K. W., Giles, K. M., & Dallas, P. B.
- Affiliations
- The Kids Research Institute Australia (AU)
- The University of Western Australia (AU)
- Corresponding author
- Dallas, Peter B
- Venue
- BMC Research Notes
- Published
- 2012-09-14
- Volume
- 5
- Issue
- 1
- Pages
- 507-507
- ISSN
- 1756-0500 (ISSN-L)
- PMC
- PMC3531299
- OpenAlex
- W1985551528
- Cited by
- 23
- References
- 65
- Citations by year
- 2025: 1 · 2021: 1 · 2020: 2 · 2018: 2 · 2017: 2 · 2016: 2 · 2015: 4 · 2014: 2 · 2013: 6 · 2012: 1
- FWCI
- 1.67
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- BMC Research Notes (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- UWA Profiles and Research Repository (UWA) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
- Keywords
- medulloblastoma, miRNA expression profiling, endogenous control genes, TaqMan Low Density Array, neural progenitor cells, small nucleolar RNA, childhood brain tumours, geNorm, NormFinder
- MeSH terms
- AC133 Antigen, Analysis of Variance, Calibration, Cell Line, Cerebellar Neoplasms, Child, Child, Preschool, Female, Glycoproteins, Humans, Infant, Male, Medulloblastoma, Peptides, Quality Control, Reference Standards, Software, Biomarkers, Tumor, Reproducibility of Results, Antigens, CD, Spheroids, Cellular, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling, MicroRNAs, Neural Stem Cells, Real-Time Polymerase Chain Reaction
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