MIQE challenges and solutions – Exosome isolation and holistic expression profiling using small RNA-Seq and RT-qPCR
Michael Pfaffl,
Professor,
Technical University of Munich
Exosomes are cell-derived vesicles that are present in many tissues and in all biological fluids, including blood, milk, urine, and sweat. Exosomes are proposed to have specialized functions and play a key role in cell-to-cell ‘hormone like ’communication, to regulate cell differentiation, cell proliferation, immune response or patho-physiological processes. Consequently, there is a growing interest in molecular diagnostics to evaluate the prognostic or clinical benefits. Very less information is available about the exosomes and their small RNA composition in biofluids such as milk and whether milk possesses its own defined microRNA profile. MicroRNAs in particular, as major content of exosomes, regulate gene expression by post transcriptional binding and thereby suppressing protein translation. They play an important role in almost all physiological or regulative processes.
The first part of the talk will cover the importance and practical considerations of the MIQE guidelines amplifying microRNAs, in particular focused on RNA integrity, PCR efficiency correction and normalisation strategies using validated reference genes.
The second part focuses on the discovery of ‘transcriptional biomarkers’ which are used across various diagnostic disciplines at a greater extent. Gene expression is a dynamic process that adapts very fast to physiological changes or exogenous stimuli and thus the transcriptome with its enormous number of alternative spliced RNAs reflects the current physiological status. Therefore monitoring the transcriptome is a potential and very promising way for detecting ‘transcriptional biomarkers’ for specific physiological situations, diseases or treatments. Herein we want to focus on expressed biomarkers discovered in the non-coding transcriptome using RT-qPCR and small RNA Seq. To generate a holistic overview of all present small RNA transcriptome RNA Seq was performed on whole blood, whole milk and exosomes in milk and blood. Exosomes were purifie
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