Introduction Breast cancer is a heterogeneous disease encompassing a number of phenotypically diverse tumours. Expression levels of the oestrogen, progesterone and HER2/ neu receptors which characterize clinically distinct breast tumours have been shown to change during disease progression and in response to systemic therapies. Mi(cro)RNAs play critical roles in diverse biological processes and are aberrantly expressed in several human neoplasms including breast cancer, where they function as regulators of tumour behaviour and progression. The aims of this study were to identify miRNA signatures that accurately predict the oestrogen receptor (ER), progesterone receptor (PR) and HER2/neu receptor status of breast cancer patients to provide insight into the regulation of breast cancer phenotypes and progression.

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.

The discovery of microRNAs (miRNA) as novel modulators of gene expression has resulted in a rapidly expanding repertoire of molecules in this family, as reflected in the concomitant expansion of scientific literature. MiRNAs are a category of naturally occurring RNA molecules that play important regulatory roles in plants and animals by targeting mRNAs for cleavage or translational repression. Characteristically, miRNAs are noncoding, single-stranded short (18-22 nucleotides) RNAs, features which possibly explain why they had not been intensively investigated until recently. Accumulating experimental evidence indicates that miRNAs play a pivotal role in many cellular functions via the regulation of gene expression. Furthermore, their dysregulation and/or mutation has been shown in carcinogenesis. We provide a brief review of miRNA biogenesis and discuss the technical challenges of modifying experimental techniques to facilitate the identification and characterization of these small RNAs. MiRNA function and their involvement in malignancy, particularly their putative role as oncogenes or tumor suppressors is also discussed, with a specific emphasis on breast cancer. Finally, we comment on the potential role of miRNAs in breast cancer management, particularly in improving current prognostic tools and achieving the goal of individualized cancer treatment.

Background: Real-time quantitative PCR (RQ-PCR) forms the basis of many breast cancer biomarker studies and novel prognostic assays, paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC) genes, used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (ESR1) was used a target gene to compare the effect of choice of EC on the estimate of gene quantity.

A purpose-designed microarray platform (Stressgenes, Phase 1) was utilised to 21 investigate the changes in gene expression within the liver of rainbow trout during 22 exposure to a prolonged period of confinement. Tissue and blood samples were collected 23 from trout at intervals up to 648 h after transfer to a standardised confinement stressor, 24 together with matched samples from undisturbed control fish. Plasma ACTH, cortisol, 25 glucose and lactate were analysed to confirm that the neuroendocrine response to 26 confinement was consistent with previous findings and to provide a phenotypic context to 27 assist interpretation of gene expression data. Liver samples for suppression subtractive 28hybridization (SSH) library construction were selected from within the experimental 29 groups comprising "early" stress (2-48h) and "late" stress (96-504h). In order to reduce 30 redundancy within the four SSH libraries and yield a higher number of unique clones an 31 additional subtraction was carried out. After printing of the arrays a series of 55 32 hybridisations were executed to cover 6 time-points. At 2h, 6h, 24h, 168h and 504h 5 33 individual confined fish and 5 individual control fish were used with control fish only at 34 0h. A preliminary list of 314 clones considered differentially regulated over the complete 35 time course was generated by a combination of data analysis approaches and the most 36 significant gene expression changes were found to occur during the 24h to 168h time 37 period with a general approach to control levels by 504h. Few changes in expression were 38 apparent over the first 6h. The list of genes whose expression was significantly 39 altered comprised predominantly genes belonging to the biological process category 40

. Interactions between the estrogen receptor, its cofactors and microRNAs in breast cancer.

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