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

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.

Figure 1

Quantification cycle (Cq) values for the technical replicates of proposed EC reference genes of the (TLDA) cards A (left) and B (right).

Figure 2

Quantification cycle (Cq) values for technical replicates of (A) MammU6 (B) RNU44 and (C) RNU48 across all samples for TaqMan low density array (TLDA) cards A and B.

Figure 3

Variation in expression of all candidate EC reference genes of (TLDA) card A and B identified as uniformly expressed between primary medulloblastoma (MB) specimens and CD133+ NSCs/ CD133- NPCs. Quantification cycle (Cq) values for candidate EC reference genes were plotted for each sample. Cq values were also plotted for RNU48, as although differentially expressed, it did not reach statistical significance.

Figure 4

Quantitative differences in miRNA expression in medulloblastoma (MB) normalising to different EC reference genes. The mean fold changes of hsa-miR-923, hsa-miR-144* and hsa-miR-21* expression in nine primary MB specimens in comparison to the mean expression of these miRNAs in CD133+ NSCs and CD133- NPCs. Relative expression was determined using the 2^-ΔCq method and log₂ transformed. Normalisation was carried out either to hsa-miR-425* (dark blue), RNU43 (red), hsa-miR-877 (green), RNU24 (purple) or geometric mean of hsa-miR-425* and RNU24 (light blue). The fold changes of all three miRNAs normalised against RNU43 was different from the fold changes obtained following the normalisation against hsa-miR-425*, hsa-miR-877, RNU24 and the geometric mean of hsa-miR-425* and RNU24.