Visualization and quantitation of the expression of microRNAs and their target genes in neuroblastoma single cells using imaging cytometry

Eugene D Ponomarev,Natasha S Barteneva,Tatiana Veremeyko

doi:10.1186/1756-0500-4-517

Eugene D Ponomarev, Natasha S Barteneva + Show 1 more

Open Access

https://doi.org/10.1186/1756-0500-4-517

Copy DOI

Abstract

BackgroundMicroRNAs (miRNAs) are regulatory molecules that play an important role in many physiological processes, including cell growth, differentiation, and apoptosis. In addition to modulating normal cellular functions, it has also been reported that miRNAs are involved in the development of many pathologies, including cardiovascular diseases, cancer, inflammation, and neurodegeneration. Methods for the sensitive detection and measurement of specific miRNAs and their cellular targets are essential for both basic research endeavours, as well as diagnostic efforts aimed at understanding the role of miRNAs in disease processes.FindingsIn this study, we describe a novel, imaging cytometry-based protocol that allows for simultaneous visualisation and quantification of miRNAs and their putative targets. We validated this methodology in a neuronal cell line by examining the relationship of the miRNA miR-124 and its known target, cyclin dependent kinase 6 (CDK6). We found that ectopic overexpression of miR-124 resulted in the downregulation of CDK6, decreased cellular proliferation, and induced cellular morphological changes.ConclusionsThis method is suitable for analysing the expression and cellular localisation of miRNAs and target proteins in small cell subsets within a heterogeneous cell suspension. We believe that our cytometry-based methodology will be easily adaptable to miRNA studies in many areas of biomedical research including neuroscience, stem cell biology, immunology, and oncology.

Highlights

MicroRNAs are regulatory molecules that play an important role in many physiological processes, including cell growth, differentiation, and apoptosis
Expression of miR-124 is inversely correlated with the expression of cyclin dependent kinase 6 (CDK6) We initially developed techniques for single staining of CDK6 or miR-124
The hybridisation conditions for the miR-124 probe included the presence of formamide in the hybridisation buffer and high temperature (53°C) which likely resulted in the denaturation of CDK6 and substantially decreased antibody staining

Summary

Introduction

MicroRNAs (miRNAs) are regulatory molecules that play an important role in many physiological processes, including cell growth, differentiation, and apoptosis. QPCR is a sensitive and quantitative method, it does not allow for the measurement of miRNA levels in specific cell subsets within a heterogeneous cell population, nor does it allow for the visualisation of specific miRNA species in particular cellular compartments of sorted cells Other methods, such as in situ hybridisation, are capable of assessing miRNA expression in specific cell types within tissue sections, but this method is not entirely quantitative, as either semiquantitative western blotting or a recently developed quantitative TaqMan Protein Assay are used to measure target protein levels in the entire cell population rather than in individual cells [9]. Quantitative imaging cytometry with the ImageStream system allows for quantitative evaluation of internalisation, co-localisation, and trafficking of proteins in various cellular compartments [11,12,13] and is a method of choice due to its ability to combine morphometric analysis of images with the statistical analysis of a large number of cells (reviewed by Zuba-Surma et al [14].)

Methods

Results

Conclusion