MIQE-Compliant Validation of MicroRNA Biomarker Signatures Established by Small RNA Sequencing.

MicroRNAs (miRNAs), a class of small non-coding RNAs that modulate gene expression at the post-transcriptional level, are attractive targets in many academic and diagnostic applications. Among them, assessing miRNA biomarkers in minimally invasive liquid biopsies was shown to be a promising tool for managing diseases, particularly cancer. The initial screening of disease-relevant transcripts is often performed by high-throughput next-generation sequencing (NGS), in here RNA sequencing (RNA-Seq). After complex processing of small RNA-Seq data, differential gene expression analysis is performed to evaluate miRNA biomarker signatures. To ensure experimental validity, biomarker candidates are commonly validated by an orthogonal technology such as reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). This chapter outlines in detail the material and methods one can apply to reproducibly identify miRNA biomarker signatures from blood total RNA. After screening miRNA profiles by small RNA-Seq, resulting data is validated in compliance with the "Minimum Information for Publication of Quantitative Real-Time PCR Experiments" (MIQE) guidelines.