Magnetic Alignment of Microelements Containing Cultured Neuronal Networks for High-Throughput Screening
High-throughput screening (HTS) on neurons presents unique difficulties because they are postmitotic, limited in supply, and challenging to harvest from animals or generate from stem cells. These limitations have hindered neurological drug discovery, leaving an unmet need to develop cost-effective technology for HTS using neurons. Traditional screening methods use up to 20,000 neurons per well in 384-well plates. To increase throughput, we use "microraft" arrays, consisting of 1600 square, releasable, paramagnetic, polystyrene microelements (microrafts), each providing a culture surface for 500–700 neurons. These mic...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Gordon, K. R., Wang, Y., Allbritton, N. L., Taylor, A. M. Tags: Original Research Source Type: research
Phenotypic Screening for Friedreich Ataxia Using Random shRNA Selection
Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix and regulates the iron–sulfur cluster (ISC) assembly complex. ISCs are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain. Decreased expression of frataxin is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute t...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Cotticelli, M. G., Acquaviva, F., Xia, S., Kaur, A., Wang, Y., Wilson, R. B. Tags: Original Research Source Type: research
Multiparametric Phenotypic Screening System for Profiling Bioactive Compounds Using Human Fetal Hippocampal Neural Stem/Progenitor Cells
In this study, we established a multiparametric screening system to detect bioactive compounds affecting the cell fate of human neural stem/progenitor cells (NSCs/NPCs), using human fetal hippocampal NSCs/NPCs, HIP-009 cells. We examined effects of 410 compounds, which were collected based on mechanisms of action (MOAs) and chemotypes, on HIP-009’s cell fate (self-renewal, neuronal and astrocytic differentiation) and morphology by automated multiparametric assays and profiled induced cellular phenotypes. We found that this screening classified compounds with the same MOAs into subgroups according to additional pharma...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Tabata, Y., Murai, N., Sasaki, T., Taniguchi, S., Suzuki, S., Yamazaki, K., Ito, M. Tags: Original Research Source Type: research
Neuronal and Cardiovascular Potassium Channels as Therapeutic Drug Targets: Promise and Pitfalls
Potassium (K+) channels, with their diversity, often tissue-defined distribution, and critical role in controlling cellular excitability, have long held promise of being important drug targets for the treatment of dysrhythmias in the heart and abnormal neuronal activity within the brain. With the exception of drugs that target one particular class, ATP-sensitive K+ (KATP) channels, very few selective K+ channel activators or inhibitors are currently licensed for clinical use in cardiovascular and neurological disease. Here we review what a range of human genetic disorders have told us about the role of specific K+ channel ...
Source: Journal of Biomolecular Screening - September 18, 2015 Category: Molecular Biology Authors: Humphries, E. S. A., Dart, C. Tags: Review Article Source Type: research
A Perspective on the Future of High-Throughput RNAi Screening: Will CRISPR Cut Out the Competition or Can RNAi Help Guide the Way?
For more than a decade, RNA interference (RNAi) has brought about an entirely new approach to functional genomics screening. Enabling high-throughput loss-of-function (LOF) screens against the human genome, identifying new drug targets, and significantly advancing experimental biology, RNAi is a fast, flexible technology that is compatible with existing high-throughput systems and processes; however, the recent advent of clustered regularly interspaced palindromic repeats (CRISPR)-Cas, a powerful new precise genome-editing (PGE) technology, has opened up vast possibilities for functional genomics. CRISPR-Cas is novel in it...
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Taylor, J., Woodcock, S. Tags: Articles Source Type: research
High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has been seized upon with a fervor enjoyed previously by small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies and has enormous potential for high-throughput functional genomics studies. The decision to use this approach must be balanced with respect to adoption of existing platforms versus awaiting the development of more "mature" next-generation systems. Here, experience from siRNA and shRNA screening plays an important role, as issues such as targeting efficiency, pooling strategies, and off-target effects with those tec...
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Wade, M. Tags: Articles Source Type: research
Targeting Human Long Noncoding Transcripts by Endoribonuclease-Prepared siRNAs
We present a compendium of lncRNA expression data for 11 human cancer cell lines. Furthermore, we show that the resource is suitable for combined knockdown and localization analysis. We discuss challenges in sequence annotation of lncRNAs with respect to their often low and cell type–specific expression and specify esiRNAs that are suitable for targeting lncRNAs in commonly used human cell lines. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Theis, M., Paszkowski-Rogacz, M., Weisswange, I., Chakraborty, D., Buchholz, F. Tags: Articles Source Type: research
MicroRNA Screening and the Quest for Biologically Relevant Targets
This article provides an overview of cell-based screenings for miRNA function that were performed in different biological contexts. The advantages and limitations of computational and experimental approaches commonly used to identify miRNA targets are also discussed. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Eulalio, A., Mano, M. Tags: Articles Source Type: research
iScreen: Image-Based High-Content RNAi Screening Analysis Tools
High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discove...
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Zhong, R., Dong, X., Levine, B., Xie, Y., Xiao, G. Tags: Articles Source Type: research
A Multivariate Computational Method to Analyze High-Content RNAi Screening Data
High-content screening (HCS) using RNA interference (RNAi) in combination with automated microscopy is a powerful investigative tool to explore complex biological processes. However, despite the plethora of data generated from these screens, little progress has been made in analyzing HC data using multivariate methods that exploit the full richness of multidimensional data. We developed a novel multivariate method for HCS, multivariate robust analysis method (M-RAM), integrating image feature selection with ranking of perturbations for hit identification, and applied this method to an HC RNAi screen to discover novel compo...
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Rameseder, J., Krismer, K., Dayma, Y., Ehrenberger, T., Hwang, M. K., Airoldi, E. M., Floyd, S. R., Yaffe, M. B. Tags: Articles Source Type: research
Versatile Viral Vector Strategies for Postscreening Target Validation and RNAi ON-Target Control
Our approach aims to optimize postscreening target validation strategies using viral vector–driven RNA interference (RNAi) cell models. The RNAiONE validation platform is an array of plasmid-based expression vectors that each drives tandem expression of the gene of interest (GOI) with one small hairpin RNA (shRNA) from a set of computed candidate sequences. The best-performing shRNA (>85% silencing efficiency) is then integrated in an inducible, all-in-one lentiviral vector to transduce pharmacologically relevant cell types that endogenously express the GOI. VariCHECK is used subsequently to combine the inducible ...
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Christel, C. J., Schmied, P., Jagusch, V., Schrodel, S., Thirion, C., Schmitt, K., Salomon, M. Tags: Articles Source Type: research
The Power Decoder Simulator for the Evaluation of Pooled shRNA Screen Performance
RNA interference screening using pooled, short hairpin RNA (shRNA) is a powerful, high-throughput tool for determining the biological relevance of genes for a phenotype. Assessing an shRNA pooled screen’s performance is difficult in practice; one can estimate the performance only by using reproducibility as a proxy for power or by employing a large number of validated positive and negative controls. Here, we develop an open-source software tool, the Power Decoder simulator, for generating shRNA pooled screening experiments in silico that can be used to estimate a screen’s statistical power. Using the negative b...
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Stombaugh, J., Licon, A., Strezoska, Z., Stahl, J., Anderson, S. B., Banos, M., van Brabant Smith, A., Birmingham, A., Vermeulen, A. Tags: Articles Source Type: research
Acoustic Liquid Handling for Rapid siRNA Transfection Optimization
In this study, we describe a methodology to utilize the flexibility and low-volume range of the Echo acoustic liquid handler to rapidly screen a matrix of transfection conditions. The matrix includes six different transfection lipids from three separate vendors across a broad range of concentrations. Our results validate acoustic liquid transfer for the delivery of siRNAs and transfection reagents. Finally, this methodology is applied to rapidly optimize transfection conditions across many tissue culture cell lines derived from various originating tissues. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Xiao, A. S., Lightcap, E. S., Bouck, D. C. Tags: Articles Source Type: research
RNAi Screening with Self-Delivering, Synthetic siRNAs for Identification of Genes That Regulate Primary Human T Cell Migration
This study therefore demonstrates the ease and benefits of conducting siRNA library screens in primary human T cells using self-delivering, chemically modified siRNAs, and it emphasizes the feasibility and potential of this approach for elucidating the signaling pathways that regulate T cell function. (Source: Journal of Biomolecular Screening)
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Freeley, M., Derrick, E., Dempsey, E., Hoff, A., Davies, A., Leake, D., Vermeulen, A., Kelleher, D., Long, A. Tags: Articles Source Type: research
Electroporation Knows No Boundaries: The Use of Electrostimulation for siRNA Delivery in Cells and Tissues
The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and...
Source: Journal of Biomolecular Screening - August 19, 2015 Category: Molecular Biology Authors: Luft, C., Ketteler, R. Tags: Articles Source Type: research
