Holistic and Affordable Analyses of MicroRNA Expression Profiles Using Tagged cDNA Libraries and a Multiplex Sequencing Strategy.

Small and long noncoding RNAs (ncRNAs) are key regulators of gene expression. Variations in ncRNA expression patterns can consequently affect the control of many cellular processes. Not just since 2006, when Andrew Z Fire and Craig C Mello were jointly awarded The Nobel Prize in Physiology or Medicine for their discovery of RNA interference, great efforts were undertaken to unleash the biomedical applicability of small noncoding RNAs, in particular microRNAs. With the technological evolution of massive parallel sequencing technologies over the last years, which now are available for an increasing number of scientists, there is a demand for comprehensible and efficient workflows reliable even for unique and valuable clinical specimens. Here we describe a highly reproducible low-cost protocol for analyses of miRNA expression patterns using tagged cDNA libraries and a multiplex sequencing strategy following an Illumina-like protocol. This protocol easily allows the identification of expression differences from samples of tissues of 1-2 mm and fluids of 50-200 μL. We further provide entry points into useful computational biology applications, whose target groups explicitly involve non-bioinformaticians.