High-Throughput RNA Interference Screening: Tricks of the Trade

Nebane, N. Miranda; Ćorić, Tatjana; Whig, Kanupriya; McKellip, Sara; Woods, LaKeisha; Sosa, Melinda; Sheppard, R. C.; Rasmussen, Lynn; Bjornsti, Mary-Ann; White, E. Lucile

doi:10.1177/2211068213486786

Cited by 8 publications

(9 citation statements)

References 5 publications

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“…Other statistical criterion has been published (Strictly Standardized Mean Difference (SSMD), Receiver Operating Characteristic curve (ROC) curves, Mean + standard deviation threshold (k), etc.) for defining quality standards of RNAi screens [ 13 , 14 ]. More detailed discussion on practical aspects of establishing statistical criterion measurements that certify the readiness of an assay for screening large compound collections can be obtained from the NCBI [ 12 ].…”

Section: Assay Optimizationmentioning

confidence: 99%

“…More detailed discussion on practical aspects of establishing statistical criterion measurements that certify the readiness of an assay for screening large compound collections can be obtained from the NCBI [ 12 ]. The siRNA-based screening optimization is discussed in detail in references [ 13 , 14 ]. The assays are transferred from bench top to automated robotic systems for compound and liquid dispensing operations.…”

Section: Assay Optimizationmentioning

confidence: 99%

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Early Probe and Drug Discovery in Academia: A Minireview

Roy

2018

High-Throughput

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Drug discovery encompasses processes ranging from target selection and validation to the selection of a development candidate. While comprehensive drug discovery work flows are implemented predominantly in the big pharma domain, early discovery focus in academia serves to identify probe molecules that can serve as tools to study targets or pathways. Despite differences in the ultimate goals of the private and academic sectors, the same basic principles define the best practices in early discovery research. A successful early discovery program is built on strong target definition and validation using a diverse set of biochemical and cell-based assays with functional relevance to the biological system being studied. The chemicals identified as hits undergo extensive scaffold optimization and are characterized for their target specificity and off-target effects in in vitro and in animal models. While the active compounds from screening campaigns pass through highly stringent chemical and Absorption, Distribution, Metabolism, and Excretion (ADME) filters for lead identification, the probe discovery involves limited medicinal chemistry optimization. The goal of probe discovery is identification of a compound with sub-µM activity and reasonable selectivity in the context of the target being studied. The compounds identified from probe discovery can also serve as starting scaffolds for lead optimization studies.

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Section: Assay Optimizationmentioning

confidence: 99%

Section: Assay Optimizationmentioning

confidence: 99%

Early Probe and Drug Discovery in Academia: A Minireview

Roy

2018

High-Throughput

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“…They were then sealed with sterile aluminum foil seals and stored at −20 °C until needed for the assay. 8 When prepared this way, the library can be dispensed and frozen weeks or even years in advance of the assay itself, making the assay protocol more amenable to HTS and more convenient. Additionally, with ADE technology, low-nanoliter volumes of each siRNA are dispensed into the wells of an assay plate.…”

Section: Resultsmentioning

confidence: 99%

“…One way to reduce this variability is through equipment optimization. We showed in a previous publication 8 that certain factors should be monitored in order to achieve good assay metrics when designing an siRNA screen. Here, we focus more on one of these factors, which is the use of ADE technology for handling reagents that are not dissolved in DMSO, such as siRNA libraries.…”

Section: Introductionmentioning

confidence: 99%

Acoustic Droplet Ejection Technology and Its Application in High-Throughput RNA Interference Screening

et al. 2016

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The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte’s Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use. This includes the transfer of many biological reagents solubilized in aqueous buffers, including siRNAs. The Echo 555 is ideal for siRNA dispensing because it is accurate at low volumes and a step-down dilution is not necessary. The potential for liquid carryover and cross-contamination is eliminated, as no tips are needed. Herein, we describe the siRNA screening platform at Southern Research’s HTS Center using the ADE technology. With this technology, an siRNA library can be dispensed weeks or even months in advance of the assay itself. The protocol has been optimized to achieve assay parameters comparable to small-molecule screening parameters, and exceeding the norm reported for genomewide siRNA screens.

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“…Since this cell line does not require an additional 3D live cell staining step, we veri ed the uniformity of cell dispensing in the DSM and ASM models through measurement of the area of A549_GFP cells expressing green uorescence in the live cell state. To determine a robust and reproducible assay, a means coe cient of variation (CV) value of less than 10% is used for a good cell-based assay [31]. When analyzing the CV values of two 3D cancer spheroid models, the CV values was under the 10 % respectively.…”

Section: Discussionmentioning

confidence: 99%

3D-Hepatocellular Carcinoma (3D-HCC) Models of Diffused and Aggregated Spheroids using a 96-Pillar/Well Plate

Lee¹,

Teng²,

Son³

et al. 2021

Preprint

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Background: Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related mortality worldwide. The genetic and physiological complexity of HCC is a major barrier to the study of tumorigenesis and identification of therapeutic targets. The three-dimensional (3D) culture of cancer cells within an extracellular matrix (ECM) provides an in vitro tumor model that best recapitulates in vivo tumor pathophysiology.Methods: In the current study, we cultured cells and made spheroids by aggregating cells or diffusing cells in Matrigel attached on the tip of the 96-pillar plate. According to the initial cell position in Matrigel, two 3D-HCC cancer in-vitro models (diffused spheroid model and aggregated spheroid model) were established. These two models were applied to drug sensitivity assays to identify drug sensitivity changes. The protein expression and cytokine activation related to the drug resistance and maintenance of the physiological properties of HCC cells were analyzed in both models. This 3D culture method not only maintained the phenotype of the tumor but allowed easy high-throughput screening (HTS) due to its 96-array plate configuration.Results: The Aggregated Spheroid Model (ASM) showed higher expression of cancer markers associated with proliferation, tight junctions formation and epithelial cell identity in HCC cells, as well as cytokine factors associated with immune cell recruitment/activation, ECM regulation, cancer interaction, and angiogenesis regulation.Conclusions: Overall, the proposed ASM better recapitulates the tumor microenvironment, demonstrating the involvement of the ECM/tight junctions in the cell-to-cell signaling processes. This provides more instructive data for in vitro screening of tumor cells.

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High-Throughput RNA Interference Screening: Tricks of the Trade

Cited by 8 publications

References 5 publications

Early Probe and Drug Discovery in Academia: A Minireview

Early Probe and Drug Discovery in Academia: A Minireview

Acoustic Droplet Ejection Technology and Its Application in High-Throughput RNA Interference Screening

3D-Hepatocellular Carcinoma (3D-HCC) Models of Diffused and Aggregated Spheroids using a 96-Pillar/Well Plate

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