Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer

Pamela A Davoren,Michael J Kerin,Aoife J Lowery,Nicola Miller,Roisin E Mcneill

doi:10.1186/1471-2199-9-76

Pamela A Davoren, Michael J Kerin + Show 3 more

Open Access

https://doi.org/10.1186/1471-2199-9-76

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Abstract

The discovery of microRNAs (miRNAs) added an extra level of intricacy to the already complex system regulating gene expression. These single-stranded RNA molecules, 18–25 nucleotides in length, negatively regulate gene expression through translational inhibition or mRNA cleavage. The discovery that aberrant expression of specific miRNAs contributes to human disease has fueled much interest in profiling the expression of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive and reproducible gene expression quantitation technique which is now being used to profile miRNA expression in cells and tissues. To correct for systematic variables such as amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalised to an endogenous control (EC) gene, which ideally, is stably-expressed across the test sample set. A universal endogenous control suitable for every tissue type, treatment and disease stage has not been identified and is unlikely to exist, so, to avoid introducing further error in the quantification of expression data it is necessary that candidate ECs be validated in the samples of interest. While ECs have been validated for quantification of mRNA expression in various experimental settings, to date there is no report of the validation of miRNA ECs for expression profiling in breast tissue. In this study, the expression of five miRNA genes (let-7a, miR-10b, miR-16, miR-21 and miR-26b) and three small nucleolar RNA genes (RNU19, RNU48 and Z30) was examined across malignant, benign and normal breast tissues to determine the most appropriate normalisation strategy. This is the first study to identify reliable ECs for analysis of miRNA by RQ-PCR in human breast tissue.

Highlights

IntroductionThe fraction of ncRNA believed to be functional in cells was once limited to the well-characterised transfer and ribosomal RNAs. The fraction of ncRNA believed to be functional in cells was once limited to the well-characterised transfer and ribosomal RNAs
98% of the human transcriptome is nonprotein-coding RNA [1,2]
The malignant breast tumour tissues were divided into three groups depending on the patient's disease progression in the five years following removal of the primary tumour; tumours from patients who did not develop metastases, those from patients who developed bone metastases (BM, n = 7) and those from patients who developed visceral and bone metastases (VBM, n = 6)

Summary

Introduction

The fraction of ncRNA believed to be functional in cells was once limited to the well-characterised transfer and ribosomal RNAs. The fraction of ncRNA believed to be functional in cells was once limited to the well-characterised transfer and ribosomal RNAs This fraction has recently been expanded to include microRNAs (miRNAs), a class of short, singlestranded RNAs that target, through nucleotide complementarity, specific messenger RNAs, enabling them to negatively modulate gene expression. First characterised in 1993, miRNAs were initially shown to be involved in the control of developmental timing in Caenhorhabditis elegans [3]. 15 years later, 541 human miRNAs have been submitted to the most recent edition of the online miRNA sequence repository, miRBase [4,5]. A very small proportion of identified miRNAs have verified roles, in (page number not for citation purposes)

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