Unbiased RNA-Seq-driven identification and validation of reference genes for quantitative RT-PCR analyses of pooled cancer exosomes

• Published in: BMC genomics Vol. 22; no. 1; p. 27
• Main Authors: Dai, Yao, Cao, Yumeng, Köhler, Jens, Lu, Aiping, Xu, Shaohua, Wang, Haiyun
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 06.01.2021; BioMed Central; BMC
• Subjects: Biomarkers; Biomolecules; Cancer; Cancer exosome; Candidates; Cell organelles; Coefficient of variation; Datasets; Diagnostic systems; Exosomes; Exosomes - genetics; Extracellular vesicles; Gene expression; Gene sequencing; Genes; Genetic aspects; Genomics; Humans; Intracellular Signaling Peptides and Proteins; Methods; MicroRNAs - genetics; miRNA; miRNA-Seq; Neoplasms - genetics; Ovarian cancer; Physiological aspects; Polymerase chain reaction; Proteins; qRT-PCR; Real-Time Polymerase Chain Reaction; Reference gene; Reverse Transcriptase Polymerase Chain Reaction; RNA sequencing; RNA-Seq; Stability analysis; Therapeutic applications; United States; miRNA-Seq; Reference gene; RNA-Seq; Cancer exosome; qRT-PCR
• ISSN: 1471-2164; 1471-2164
• DOI: 10.1186/s12864-020-07318-y

Exosomes are extracellular vesicles (EVs) derived from endocytic compartments of eukaryotic cells which contain various biomolecules like mRNAs or miRNAs. Exosomes influence the biologic behaviour and progression of malignancies and are promising candidates as non-invasive diagnostic biomarkers or as targets for therapeutic interventions. Usually, quantitative real-time polymerase chain reaction (qRT-PCR) is used to assess gene expression in cancer exosomes, however, the ideal reference genes for normalization yet remain to be identified. In this study, we performed an unbiased analysis of high-throughput mRNA and miRNA-sequencing data from exosomes of patients with various cancer types and identify candidate reference genes and miRNAs in cancer exosomes. The expression stability of these candidate reference genes was evaluated by the coefficient of variation "CV" and the average expression stability value "M". We subsequently validated these candidate reference genes in exosomes from an independent cohort of ovarian cancer patients and healthy control individuals by qRT-PCR. Our study identifies OAZ1 and hsa-miR-6835-3p as the most reliable individual reference genes for mRNA and miRNA quantification, respectively. For superior accuracy, we recommend the use of a combination of reference genes - OAZ1/SERF2/MPP1 for mRNA and hsa-miR-6835-3p/hsa-miR-4468-3p for miRNA analyses.