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

Abstract

BackgroundMedulloblastoma (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.ResultsExpression 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.ConclusionsA 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.

U6 snRNA is a Suitable Endogenous Control for microRNA-124 and -134 in Cultured Rat Hippocampal Neurons

“…This difference is likely to be due to the different cell types used and the different experimental conditions. This may also explain the contrasting report by Genovesi et al (2012) on U6 snRNA in stem cells. Most of these studies have looked at endogenous controls in neuronal differentiation in cell lines whilst our study is focused on maturing neurons involved in synapse formation.…”

miR-218 is downregulated and directly targets SH3GL1 in childhood medulloblastoma

“…Numerous studies have demonstrated significantly altered miRNA expression patterns in types of human cancer, deregulation of miRNA expression and the contribution of miRNAs to the multistep processes of carcinogenesis either as oncogenes or as tumor-suppressor genes (8,9). A number of miRNA expression profiling studies have demonstrated that miRNA expression is dysregulated, by the comparison of MBs with normal cerebellum (10)(11)(12). A previous study determined that the expression of miR-17~92 was upregulated in MBs and promoted cell proliferation (13).…”

Comparison of different normalization strategies for the analysis of glomerular microRNAs in IgA nephropathy

“…M -values were higher than expected (0.8–1.2) and they were even ranked differently on card A and card B. This might be due to intercard variability of snoRNAs as described by others37. In mRNA studies M -values should be less than 0.520.…”