Abstract 5280: Identification of serum circulating non-coding RNAs as diagnostic markers by Next Generation Sequencing (NGS) and low-density array.
Authors: Montani Francesca , Carletti Rose Mary , Marzi Matteo J. , Bucci Gabriele , Nicassio Francesco , Di Fiore Pier Paolo 
Published: August 2014

Abstract
Background. High incidence and mortality rate tumours present a more favourable prognosis if diagnosed in early stages (Jemal A. et al, 2011). Screening programs based on effective radiological techniques (e.g. mammography or low dose spiral CT) promise a mortality reduction, but their application on large scale suffers of disadvantages such as the use of ionizing radiation, overdiagnosis risk and high costs. Thus, the identification of molecular markers for cancer early diagnosis, especially if detectable through a simple non-invasive blood test, would provide powerful tools for the improvement of screening programs. Recently, a novel class of small non-coding RNAs, namely microRNAs, has been identified. MiRNA are extremely stable in biological fluids and their levels are different in malignant vs. control sera, making them a promising class of biological markers for early diagnosis. Indeed, studies from our and other labs led to the identification of “miRNA signatures” able to predict the presence of tumours even within a population of asymptomatic high-risk individuals (Bianchi F. et al., 2011; Boeri M. et al., 2011). However, methods used in these studies rely on QPCR approaches, which require previous knowledge on the molecules to be investigated. Indeed, the real complexity of circulating RNAs is still obscure, comprising other classes of molecules not yet investigated that could behave as novel biomarkers. The recent development of high-throughput sequencing technologies (Next Generation Sequencing - NGS) provided instruments to reveal the complexity of nucleic acids, but a complete protocol for the identification of circulating RNAs has not been established nor compared with current available approaches (i.e. QPCR).
Aim: Our aim is to develop a simultaneous and comparative protocol for serum miRNA analysis, which encompasses both high-throughput QPCR and sequencing analysis in order to reveal the complexity of circulating small-RNAs.
Methods: Total RNA has been isolated from the serum of healthy donors and analysed simultaneously by low-density QPCR (TaqMan Low Density Array, Life Technologies) and by NGS (Illumina).
Results: A step-wise protocol to simultaneously analyze non-coding RNAs using NGS technology (Illumina) and high-throughput QPCR has been developed. The protocol has been optimized to allow both analyses using even limited amount of serum (up to 1mL). Both platforms resulted as highly efficient and quantitative, although NGS manages to score many molecules and RNA species that could not be analysed by current QPCR platforms, thus revealing a major complexity of circulating non-coding RNAs.
Conclusion: We proposed that the combined use of NGS and QPCR platforms would allow a wider and more detailed analysis of circulating RNAs, expanding our ability to fish out robust and efficient molecular markers for early detection of cancer.
Citation Format: Francesca Montani, Rose Mary Carletti, Matteo J. Marzi, Gabriele Bucci, Francesco Nicassio, Pier Paolo Di Fiore. Identification of serum circulating non-coding RNAs as diagnostic markers by Next Generation Sequencing (NGS) and low-density array. [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 5280. doi:10.1158/1538-7445.AM2013-5280