In Vivo Interrogation of the Hypoxic Transcriptome of Solid Tumors: Optimizing Hypoxic Probe Labeling with Laser Capture Microdissection for Isolation of High-Quality RNA for Deep Sequencing Analysis.
Global gene expression analysis is a powerful method for identifying biological networks and regulatory mechanisms that govern cellular or tissue-level responses to physiologic stress. In the context of tumor biology, differential gene expression studies have provided information about the growth, aggressiveness, prognosis, and therapeutic response of tumors in patients. Scientists are using these valuable data to investigate pathways that can be targeted therapeutically with the goal of improving patient outcome. RNA sequencing enables nucleotide resolution of expression of whole transcriptomes, but arrives with a new set of challenges surrounding the management and analysis of large datasets. This chapter aims to review technical advancements to current methods for isolating high-quality RNA for sequencing studies directly from hypoxic tissues and introduces select widely used applications for gene expression analyses of next-generation sequencing data.
Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.|Cellular and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Lauren K
LK
Brady
Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Vladimir
V
Popov
Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. Costas.Koumenis@uphs.upenn.edu.
Constantinos
C
Koumenis
D015687:Cell Hypoxia / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D061346:Laser Capture Microdissection / Q000379:methods*; D015335:Molecular Probes / Q000378:metabolism*; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology*; D012313:RNA / Q000302:isolation & purification*; D013194:Staining and Labeling; D013223:Statistics as Topic; D059467:Transcriptome / Q000235:genetics*
D015335:Molecular Probes; D012313:RNA
Gene expression; Hypoxia; Laser capture microdissection; RNA sequencing; Tumor microenvironment