Identification of suitable endogenous control genes for microRNA expression profiling of childhood medulloblastoma and human neural stem cells

Genovesi, Laura A., Anderson, Denise, Carter, Kim W., Giles, Keith M. and Dallas, Peter B. (2012) Identification of suitable endogenous control genes for microRNA expression profiling of childhood medulloblastoma and human neural stem cells. BMC Research Notes, 5 507.1-507.12. doi:10.1186/1756-0500-5-507


Author Genovesi, Laura A.
Anderson, Denise
Carter, Kim W.
Giles, Keith M.
Dallas, Peter B.
Title Identification of suitable endogenous control genes for microRNA expression profiling of childhood medulloblastoma and human neural stem cells
Journal name BMC Research Notes   Check publisher's open access policy
ISSN 1756-0500
Publication date 2012
Sub-type Article (original research)
DOI 10.1186/1756-0500-5-507
Open Access Status DOI
Volume 5
Start page 507.1
End page 507.12
Total pages 12
Place of publication London, UK
Publisher BioMed Central
Language eng
Formatted abstract
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
Keyword Gene expression profiling
Medulloblastoma
MicroRNA
Neural stem cells
Quantitative RT-PCR
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
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Created: Tue, 20 May 2014, 14:51:12 EST by Laura Genovesi on behalf of Institute for Molecular Bioscience