Abstract 1940: High-throughput imaging and high content analysis of disease relevant lncRNAs examined by RNA fluorescence in situ hybridization (RNA FISH).
Authors: Johansson Hans E. , Orjalo Arturo V. , Coassin Sally R. , Ruth Jerry. L. , Stossi Fabio , Mancini Michael A. 
Published: August 2014

Abstract
The genome is extensively transcribed, yet more than 50 % of RNA transcripts lack protein coding potential. In recent years, long noncoding RNAs (lncRNAs) have been shown to play extensive roles in all the central aspects of gene function including imprinting, epigenetic regulation, transcription, splicing and nuclear/cytoplasmic trafficking, translation and RNA stability, with wide ranging effects on cell cycle and differentiation. Due to the increasingly central role of lncRNAs in gene transcription and disease states, it is important to develop tools to allow direct visualization and quantification of lncRNAs that could be useful both in the basic science arena and in the clinical setting with possible uses both in diagnostics and prognostics.
We recently streamlined both the design and synthesis of fluorescently labeled probe sets containing multiple tiled 20-mer GC-balanced oligonucleotides for use in RNA fluorescence in situ hybridization (RNA FISH). In contrast to traditional cell-disruptive RNA analyses (e.g. northern blotting and qPCR) that report relative population averages of RNA content, single molecule RNA FISH affords specific and sensitive detection of RNAs and quantitatively establishes the intracellular localization of the target lncRNAs.
We have validated an imaging based platform to perform RNA FISH in both manual (coverslip) and automated (384 well) formats that allows tremendous multiplexing and throughput using high throughput microscopy. Moreover, we developed high content analysis routines that permit quantification of RNA particles at the single cell level and determine their localization and pattern (texture). As specific lncRNAs (e.g. MALAT1) are known to target specific intranuclear domains (e.g. splicing speckles) we developed classification methods based upon sets of intensity and texture features to compare intracellular patterns of novel/less characterized ncRNAs. Moreover, this framework will also permit to address changes in lncRNA expression and patterns during the cell cycle since large number of cells can be analyzed simultaneously. We are now expanding our analysis to primary tumor tissues where lncRNAs patterns will be correlated with known FISH-compatible biomarkers, thus providing a picture simultaneously for intra- and inter-cell variation.
In summary, the results and tools presented here will enable the study of disease related lncRNAs in a high content, high throughput mode. These efforts will enable gaining better understanding of how expression and localization of lncRNAs may contribute to the diseased phenotype, and add a large selection of novel biomarkers that can be interrogated for improved diagnosis, prognosis, and therapeutic choice.
Citation Format: Hans E. Johansson, Arturo V. Orjalo, Sally R. Coassin, Jerry. L. Ruth, Fabio Stossi, Michael A. Mancini. High-throughput imaging and high content analysis of disease relevant lncRNAs examined by RNA fluorescence in situ hybridization (RNA FISH). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1940. doi:10.1158/1538-7445.AM2013-1940