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. 2023:2650:65-75.
doi: 10.1007/978-1-0716-3076-1_6.

TORNADO-seq: A Protocol for High-Throughput Targeted RNA-seq-Based Drug Screening in Organoids

Maxim Norkin  1 , Joerg Huelsken  2
Affiliations

TORNADO-seq: A Protocol for High-Throughput Targeted RNA-seq-Based Drug Screening in Organoids

Maxim Norkin et al. Methods Mol Biol. 2023.

Abstract

Organoids are 3D ex vivo cell aggregates derived from primary tissue and shown to closely recapitulate tissue homeostasis. Organoids deliver certain advantages compared to 2D cell lines and mouse models, especially in drug-screening studies and translational research projects. The application of organoids in the research field is fast-emerging and new techniques for organoid manipulation are constantly developing. Despite recent advances, RNA-seq-based drug-screening platforms in organoids are not yet established. Here, we provide a detailed protocol for performing TORNADO-seq, a targeted RNA-seq-based drug-screening method in organoids. Analyzing complex phenotypes with a large number of carefully selected read-outs allows to directly classify and group drugs even without structural similarity or overlapping mode of actions from prior knowledge. Our assay principle combines cost-effectiveness and sensitive detection of multiple cell identities, signaling pathways, and key drivers of cellular phenotypes and can be applied to many systems where this new form of high-content screening can provide information not obtainable otherwise.

Keywords: High-content screening; High-throughput screening; NGS-based screening in organoids; Targeted RNA-seq organoids.

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References

    1. Barcellos-Hoff MH, Aggeler J, Ram TG, Bissell MJ (1989) Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 105:223–235. https://doi.org/10.1242/dev.105.2.223 - DOI - PubMed
    1. Petersen OW, Rønnov-Jessen L, Howlett AR, Bissell MJ (1992) Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci U S A 89:9064–9068. https://doi.org/10.1073/pnas.89.19.9064 - DOI - PubMed - PMC
    1. Bissell MJ, Kenny PA, Radisky DC (2005) Microenvironmental regulators of tissue structure and function also regulate tumor induction and progression: the role of extracellular matrix and its degrading enzymes. Cold Spring Harb Symp Quant Biol 70:343–356. https://doi.org/10.1101/sqb.2005.70.013 - DOI - PubMed - PMC
    1. Simian M, Bissell MJ (2017) Organoids: a historical perspective of thinking in three dimensions. J Cell Biol 216:31–40. https://doi.org/10.1083/jcb.201610056 - DOI - PubMed - PMC
    1. Sato T, Vries RG, Snippert HJ et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265. https://doi.org/10.1038/nature07935 - DOI - PubMed

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