Functional characterizations and molecular dissections of long noncoding RNAs (lncRNAs) are critical to understand their involvement in the cellular regulatory network.  https://www.selleckchem.com/products/2-Methoxyestradiol(2ME2).html  LncRNAs exert their effects through functional RNA domains that interact with other molecules, including proteins, DNA, and RNA. Here, we describe experimental procedures for generating genomic deletions in a human haploid cell line using the CRISPR/Cas9 system. This method can be applied to examine functions of lncRNAs and their domains by establishing knockout and partial deletion mutant cell lines. In addition, we describe a CRISPR-mediated knockin method for artificial tethering of partner RNA-binding proteins to lncRNAs and its use to validate lncRNA-mediated functions.With the rapid revolution in RNA/DNA sequencing technologies, it is evident that mammalian genomes express tens of thousands of long noncoding RNAs (lncRNAs). Since a large majority of lncRNAs have been functionally implicated in cancer development and progression, there is an increasing appreciation for the use of antisense oligonucleotide (ASO)-based therapies targeting lncRNAs in several cancers. Despite their great potential in therapeutic applications, their use is still limited due to cellular toxicity and shortcomings in achieving required stability in biological fluids and tissue uptake. To overcome these limitations, major changes in ASO chemistry have been introduced to generate second and third generation ASOs, including locked nucleic acids (LNA) technology. Here we describe two different LNA-ASO delivery approaches, a peritumoral administration and a systemic delivery in xenograft models of lung adenocarcinoma, that significantly reduced tumor growth without inducing toxicity.The systematic investigation of RNA-protein interactions is a key step towards a better understanding of the functions of RNA molecules.We developed an easy-to-use method to isolate and identify RNAs and proteins bound to long non-coding RNAs (lncRNAs ) in their native configuration. Similar to other methodologies, we utilize biotinylated antisense oligonucleotides (ASOs) to purify the lncRNA of interest and its associated proteins from different cellular compartments.Immunofluorescence and fluorescence in situ hybridization (FISH) are widely used cytogenetic techniques for visualization of protein and RNA/DNA molecules. Here, we describe an experimental procedure for quick sequential immunofluorescence and RNA FISH (immuno-FISH), which enables the simultaneous detection of proteins, chromatin modifications, and RNAs on the inactive X-chromosome (Xi) using female mouse embryonic fibroblast (MEF) and tail-tip 3T3 cell lines. Using a pooled array of oligonucleotides labeled with a single fluorophore as an RNA FISH probe, we can reduce the time for RNA FISH from an overnight process to 1-2 h without losing its sensitivity. This protocol could be applied to visualization of various protein and RNA molecules, and chromatin modifications.From high-throughput DNA and RNA sequencing technologies, it is evident that more than two-thirds of the mammalian genome is transcribed and nearly 98% of the transcriptional output in humans constitute noncoding RNA, comprising tens of thousands of small and long noncoding RNAs. These observations have put the study of RNA expression levels at the center of molecular biology research. The transcriptional output of cells changes temporally throughout different cell cycle phases, or in response to a large panel of stimuli. In such instances, the measure of induced RNA transcripts might be obscured by the presence of steady-state RNA levels in the total transcriptome. With this protocol, we provide a method for labeling and purification of the nascent RNAs transcribed over short periods of time in cultured cells. The supplementation of cell culture medium with a chemically modified analog of uridine, ethynyl-uridine, allows for the subsequent biotinylation of ethynyl-uridine residues with a click-chemistry reaction. The labeled RNA is then purified on streptavidin beads and eluted. The purified RNA is suitable for use in RT-qPCR assays as well as in deep sequencing applications.DMS-MaPseq is a chemical probing method combined with high throughput sequencing used to study RNA structure. Here we present a flexible protocol for adherent and suspension mammalian cells to analyze RNA structure in vitro or in vivo. The protocol provides instruction on either a targeted sequencing of a lncRNA of interest or a transcriptome-wide approach that provides structural data on all expressed RNAs, including lncRNAs. This technique is particularly useful for comparing in vitro and in vivo structure of RNAs, determining how mutations and polymorphisms with phenotypic effects influence RNA structure and analyzing RNA structure across the entire transcriptome.Long noncoding RNAs (lncRNAs) contain >200 nucleotides and act as regulatory molecules in transcription and translation processes in both normal and pathological conditions. LncRNAs have been reported to localize in nuclei, cytoplasm, and, more recently, extracellular vesicles such as exosomes. Exosomal lncRNAs have gained much attention as exosomes secreted from one cell type can transfer their cargo (e.g., protein, RNA species, and lipids) to recipient cells and mediate phenotypic changes in the recipient cell. In recent years, many exosomal lncRNAs have been discovered and annotated and are attracting much attention as potential markers for disease diagnosis and prognosis. It is expected that many exosomal lncRNAs are yet to be identified. However, characterization of unannotated exosomal RNAs with non-protein-coding sequences from massive RNA sequencing data is technically challenging. Here, we describe a method for the discovery of annotated and unannotated exosomal lncRNA. This method includes a large-scale isolation and purification strategy for exosome subtypes, using the human colorectal cancer cell line (LIM1863) as a model. The method inputs RNA sequencing clean reads and performs transcript assembly to identify annotated and unannotated exosomal lncRNAs. Cutoffs (length, number of exon, classification code, and human protein-coding probability) are used to identify potentially novel exosomal lncRNAs. Raw read count calculation and differential expression analysis are also introduced for downstream analysis and candidate selection. Exosomal lncRNA candidates are validated using RT-qPCR. This method provides a template for exosomal lncRNA discovery and analysis from next-generation RNA sequencing.