Sweta Rani - MicroRNA Profiling: Methods and Protocols

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For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

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microRNAs (miRNAs) are small non-coding RNAs. miRNAs play a central role in regulating the expression of genes at the post-translation level. One miRNA can bind to multiple mRNAs and have multiple binding sites regulating multiple targets and pathways. Dysregulation of miRNAs is associated with several diseases and is a key regulator of several biological processes. Expression of miRNAs can be detected in tissue specimens, biological fluids, as well as cell lines and have a potential as biomarkers for therapeutical and medicinal interventions.

This is the second edition of microRNA Profiling from the Methods in Molecular Biology series. This book collates chapters contributed by experts in their respective fields. It includes both classical techniques and newer approaches to isolate and profile miRNAs. The chapters describe detailed step-by-step protocol and troubleshooting tips ensuring successful experiment.

This volume includes chapters that comprehensively describe miRNA biogenesis and its function in regulating progression of several diseases. The first few chapters describe optimised protocol of isolating RNA from various samples including exosomes, serum specimens and cell lines and determine the quality and quantity of RNA. 3D cell culture allows the cells to interact with their surroundings in all the three dimensions. Two of the chapters in this book describe the 3D techniques used to culture cells and ways to transfect miRNAs.

Exosomes or extracellular vesicles (EVs) are secreted by almost all cell types and are one of the mechanisms of intercellular communication. EVs are known to contain not only RNAs, proteins, lipids and metabolites but also miRNAs. miRNAs encapsulated in these EVs are protected from degradation by plasma ribonucleases. Circulating miRNAs hold immense potential as diagnostic, prognostic and/or predictive biomarkers. Some of the chapters compare and contrast various methods to profile exosomal miRNA.

The last two chapters include easy-to-follow protocol for analysing miRNA profiling data, reducing technical difficulties. Galaxy software is used to analyse genomic data including miRNA and miRDeep2 for identification and quantification of miRNA from sequencing data. miRDeep-P2 can also be used for miRNA annotation in plants using deep sequencing data.