The choice of negative control antisense oligonucleotides dramatically impacts downstream analysis depending on the cellular background.

BACKGROUND: The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism's physiological function. Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. The recent technological boost in sequencing approaches unveiled long noncoding RNAs (lncRNAs) as prominent regulatory players of various gene expression levels in a cell-type-specific manner. RESULTS: To investigate the potential roles of lncRNAs in vascular biology, we performed antisense oligonucleotide (ASO) knockdowns of lncRNA candidates specifically expressed either in human lymphatic or blood vascular endothelial cells (LECs or BECs) followed by Cap Analysis of Gene Expression (CAGE-Seq). Here, we describe the quality control steps adopted in our analysis pipeline before determining the knockdown effects of three ASOs per lncRNA target on the LEC or BEC transcriptomes. In this regard, we especially observed that the choice of negative control ASOs can dramatically impact the conclusions drawn from the analysis depending on the cellular background. CONCLUSION: In conclusion, the comparison of negative control ASO effects on the targeted cell type transcriptomes highlights the essential need to select a proper control set of multiple negative control ASO based on the investigated cell types.

Tips for Successful lncRNA Knockdown Using Gapmers.

Long noncoding RNAs (lncRNAs) are a recently discovered class of RNA that have diverse intracellular regulatory and structural roles. Because of their wide assortment of functions, lncRNAs can have varied distributions in the nucleus and/or cytoplasm of a cell. However, even though tens of thousands of human lncRNAs have been identified, currently less than 3% have empirically validated functions. RNA knockdown is now a relatively commonplace laboratory technique used to functionally characterize an RNA. These techniques (most commonly antisense therapy and RNA interference) can even have therapeutic benefit to treat a wide variety of genetic or infectious diseases as evidenced by the several RNA knockdown reagents currently in clinical trials. This protocol describes the use of validated gapmer antisense oligonucleotides (ASOs) to knockdown human MALAT1, a nuclear-retained lncRNA that is upregulated in multiple cancer cells. Methods used include cationic lipid transfection into HeLa cells, RNA isolation, and RT-qPCR analysis of the RNA knockdown levels.

Human Genome-Edited Babies: First Responder with Concerns Regarding Possible Neurological Deficits!

The ultimate outcome in genome-editing research stepped into unknown territories last month when two babies were brought into the world with clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) facilitated knockdown of chemokine receptor 5 (CCR5). An immediate outcry by the public and the scientific community followed, which is still ongoing with much apprehensions and criticism of the ethical and scientific aspects of the procedure and its effects on the future of genome editing needed in other stubborn inheritable diseases for which there is no cure at present. With the debate on the consequences of this particular receptor knockdown still going on and the after-shocks in the form of queries expected to continue for some time in the future, we enter the arena of this particular genome editing as first responders with concerns regarding the neurological aftermath of CCR5 knockout in the babies born.

Development and validation of a cell-based assay for the nuclear receptor retinoid-related orphan receptor gamma.

The nuclear receptor retinoid-related orphan receptor gamma (RORγ) has become an attractive target for drug discovery due to its important role in the development and differentiation of Th17 cells, a subset of T cells that produce interleukin-17 and are involved in the pathogenesis of human inflammatory and autoimmune diseases. To facilitate the drug discovery efforts in this area, we have developed a cellular assay for screening for RORγ inverse agonists. We stably engineered a tetracycline-inducible Gal4 DNA-binding domain/RORγ ligand-binding domain fusion protein into an upstream activation sequence driven-beta-lactamase reporter gene cell line. Due to its constitutive activity, the induced Gal4-RORγ expression leads to increased reporter activity, which can be knocked down using RORγ ligand-binding domain-specific RNA interference oligos. Using this assay, we tested several recently reported ligands for RORγ and observed varying levels of partial inverse agonist activity at µM concentrations. Additionally, we screened a small library of biologically active compounds with this assay and demonstrated its robustness and usefulness in high-throughput screening and follow-up studies for this emerging drug target.

siRNAs: their potential as therapeutic agents--Part I. Designing of siRNAs.

RNA interference (RNAi) is a novel and essential biological process, as well as a powerful experimental tool with the potential to be used in therapeutic development. RNAi-based strategies have the capability of being able to be driven from bench to bedside. It is very important to develop the precise tools for designing the siRNAs to get the most efficient knockdown of the target genes and to reduce any off-target effects. In this review we have discussed the strategies and parameters required for effective siRNA designing and synthesis, based on already published literature.

Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.

RNAi using siRNA is a very powerful tool for functional genomics to identify new drug targets and biological pathways. Although their use in epithelial cells is relatively easy and straightforward, transfection in other cell types is still challenging. The authors report the optimization of transfection conditions for Raw 267.4 macrophage cells. The herein described procedure makes use of automated confocal microscopy, enhanced green fluorescent protein (EGFP)-expressing macrophages, and fluorescently labeled siRNAs to simultaneously quantify both siRNA uptake and silencing efficiency. A comparison of 10 commercial transfectants was performed, leading to the selection of the transfectant giving the highest reproducible knock-down effect without inducing cell toxicity or cell activation. Several buffers used for siRNA/lipid complex assembly were tested, and such a study revealed the crucial importance of this parameter. In addition, a kinetics study led to the determination of the optimal siRNA concentration and the best time window for the assay. In an original approach aimed at simultaneously optimizing both the high-throughput screening process and biological factors, optimal reagent volumes and a process flowchart were defined to ensure robust silencing efficiencies during screening. Such an account should pave the way for future genome-wide RNAi research in macrophages and present an optimization procedure for other "hard-totransfect" cell lines.