Main description:

This volume aims at providing a selection of the most effective and widely employed protocols used in the Ewing sarcoma field. Chapters are divided into five sections detailing the molecular biology techniques used to analyze Ewing sarcoma including tissue preservation and methods used in morpho-molecular diagnostics and routine immune-histological analysis. Additional wet-lab protocols comprise in vitro techniques including re-expression and functional analyses of target genes together with in vivo tools and models such as patient-derived xenografts (PDX) and orthotopic xenografts for metastasis assessment in Ewing sarcoma. A full section on bioinformatic approaches to Ewing sarcoma covering ChIPseq, epigenetic and systems biology analyses finalizes this volume. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and clearly written, Ewing sarcoma: Methods and Protocols aims to provide methods to further the study of Ewing sarcoma and bring researchers one step forward in the quest to fight this devastating disease.

Contents:

Part I: Introduction

1. Germline Variation and Somatic Alterations in Ewing Sarcoma

Mitchell J. Machiela and Thomas G. P. Grunewald

2. Western Blot Analysis in Ewing Sarcoma

Aruna Marchetto and Laura Romero-Perez

Part II: Molecular Biology Techniques Applied To Ewing Sarcoma

3. Tissue Preservation and FFPE Samples: Optimized Nucleic Acids Isolation in Ewing Sarcoma

Laura Romero-Perez and Thomas G. P. Grunewald

4. Liquid Biopsies in Ewing Sarcoma

Manuela Krumbholz and Markus Metzler

5. (Immuno-)histological Analysis of Ewing Sarcoma

David Marcilla, Isidro Machado, Thomas G. P. Grunewald, Antonio Llombart-Bosch, and Enrique de Alava

6. Molecular Approaches to Diagnosis In Ewing Sarcoma: Fluorescence in situ hybridization (FISH)

Marcel Trautmann and Wolfgang Hartmann

7. Molecular Approaches to Diagnosis in Ewing Sarcoma: RT-PCR

Carlos Rodriguez-Martin and Javier Alonso

8. Molecular Approaches to Diagnosis in Ewing Sarcoma: Targeted RNA Sequencing

Carmen Salguero-Aranda and Juan Diaz-Martin

Part III: Diagnostics in Ewing Sarcoma

9. Ewing-specific (re)expression Models

Maximilian M. L. Knott and Florencia Cidre-Aranaz

10. Analysis of Regulatory Sequences by Dual-luciferase Reporter Assays in Ewing Sarcoma

Tilman L. B. Hoelting and Maximilian M. L. Knott

Part IV: Cellular biology and cell culture techniques

11. Proliferation Assessment by Trypan Blue Exclusion in Ewing Sarcoma

Cornelius Funk and Julian Musa

12. Drug Screening by Resazurin Colorimetry in Ewing Sarcoma

Julian Musa and Florencia Cidre-Aranaz

13. Analysis of Migration and Invasion in Ewing Sarcoma

Florencia Cidre-Aranaz

Part V: Animal models in Ewing sarcoma

14. Genetically Engineered Mouse Model in Ewing Sarcoma

Miwa Tanaka and Takuro Nakamura

15. Tumor Growth Analysis of Ewing Sarcoma Cell Lines Using Subcutaneous Xenografts in Mice

Florencia Cidre-Aranaz and Shunya Ohmura

16. Metastasis Assessment in Ewing Sarcoma Using Orthotopic Xenografts

Roser Lopez-Alemany and Oscar M. Tirado

17. Orthotopic Implants in Mice

Elizabeth Stewart

18. Ewing Sarcoma PDX Models

D. Surdez, L. Landuzzi, K. Scotlandi, and M.C. Manara

Part VI: Bioinformatic approaches to Ewing sarcoma 19. Using Zebrafish Larvae as a Xenotransplantation Model to Study Ewing Sarcoma Susana Pascoal, Sarah Grissenberger, Eva Scheuringer, Rita Fior, Miguel Godinho Ferreira, and Martin Distel

20. Main Repositories and Databases used in Ewing Sarcoma Research

Maria Debiec-Rychter

21. Chromatin Immunoprecipitation Followed by Next-Generation Sequencing (ChIP-seq) Analysis in Ewing Sarcoma

Gwenneg Kerdivel and Valentina Boeva

22. Epigenetic Analysis in Ewing Sarcoma

Jeremy M. Simon and Nicholas C. Gomez

23. Systems Biology Analysis for Ewing Sarcoma

Marianyela Petrizzelli, Jane Merlevede, and Andrei Zinovyev