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This volume presents techniques needed for the study of long non-coding RNAs (lncRNAs) in cancer from their identification to functional characterization. Chapters guide readers through identification of lncRNA expression signatures in cancer tissue or liquid biopsies by RNAseq, single Cell RNAseq, Phospho RNAseq or Nanopore Sequencing techniques; validation of lncRNA signatures by Real time PCR, digital PCR or in situ hybridization; and functional analysis by siRNA or CRISPR based methods for lncRNA silencing or overexpression. Lipid based nanoparticles for delivery of siRNAs in vivo, lncRNA-protein interactions, viral lncRNAs and circRNAs are also treated in this volume. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and practical, Long Non-Coding RNAs in Cancer aims to provide a collection of laboratory protocols, bioinformatic pipelines, and review chapters to further research in this vital field.
List of contents
LncRNAs and Available Databases.- LncRNA as Cancer Biomarkers.- Bioinformatics Pipeline to Analyze lncRNA Arrays.- Boinformatic Pipelines to Analyze lncRNAs RNAseq Data.- Single Cell RNAseq Analysis of lncRNAs.- Measuring lncRNA Expression by Real Time PCR.- Quantitation of Long Non-coding RNA using Digital PCR.- Detection of lncRNA by LNA-based In Situ Hybridization in Paraffin Embedded Cancer Cell Spheroids.- In vitro Silencing of lncRNA Expression using siRNAs.- In vitro silencing of lncRNAs using LNA gapmeRs.- Methods used to make Lipid Nanoparticles to deliver LNA Gapmers against lncRNAs into Acute Myeloid Leukemia (AML) Blasts.- CRISPR/Cas9 to Silence Long Non-coding RNAs.- CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) to explore the oncogenic lncRNA network.- In vivo Silencing/Overexpression of lncRNAs by CRISPR/Cas System.- Experimental Validation of the Non-Coding Potential for lncRNA.- Identification of lncRNA-protein interactions by CLIP and RNA pull-down assays.- Empirical Validation of Overlapping Virus lncRNAs and Coding Transcripts by Northern Blot.- Phospho-RNAseq profiling of extracellular mRNAs and lncRNAs.- Detection of Circulating RNA using Nanopore Sequencing.- LncRNA Quantification from Extracellular Vesicles Isolated from Blood Plasma or Conditioned Media.- The use of circRNAs as Biomarkers of Cancer.- Bioinformatic Analysis of Circular RNA Expression.- Study of circular RNAs (circRNAs) expression.
