Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

Autor(es)

Prendergast, Emily N. ^[1] ; de Souza Fonseca, Marcos Abraao ^[2] ; Dezem, Felipe Segato ^[2] ; Lester, Jenny ^[1] ; Karlan, Beth Y. ^[1] ; Noushmehr, Houtan ^{[2, 3]} ; Lin, Xianzhi ^[1] ; Lawrenson, Kate ^{[1, 4]}

Afiliação do(s) autor(es)

^[1] Cedars Sinai Med Ctr, Samuel Oschin Comprehens Canc Inst, Womens Canc Program, Los Angeles, CA 90048 - USA

^[2] Univ Sao Paulo, Dept Genet, Ribeirao Preto, SP - Brazil

^[3] Henry Ford Hlth Syst, Dept Neurosurg, Detroit, MI - USA

^[4] Cedars Sinai Med Ctr, Samuel Oschin Comprehens Canc Inst, Ctr Bioinformat & Funct Genom, Los Angeles, CA 90048 - USA

• Tipo de documento: Artigo Científico
• Fonte: PLoS One; v. 13, n. 5 MAY 8 2018.
• Citações Web of Science: 4

Resumo

Exosomes are endosome-derived membrane vesicles that contain proteins, lipids, and nucleic acids. The exosomal transcriptome mediates intercellular communication, and represents an understudied reservoir of novel biomarkers for human diseases. Next-generation sequencing enables complex quantitative characterization of exosomal RNAs from diverse sources. However, detailed protocols describing exosome purification for preparation of exosomal RNA-sequence (RNA-Seq) libraries are lacking. Here we compared methods for isolation of exosomes and extraction of exosomal RNA from human cell-free serum, as well as strategies for attaining equal representation of samples within pooled RNA-Seq libraries. We compared commercial precipitation with ultracentrifugation for exosome purification and confirmed the presence of exosomes via both transmission electron microscopy and immunoblotting. Exosomal RNA extraction was compared using four different RNA purification methods. We determined the minimal starting volume of serum required for exosome preparation and showed that high quality exosomal RNA can be isolated from sera stored for over a decade. Finally, RNA-Seq libraries were successfully prepared with exosomal RNAs extracted from human cell-free serum, cataloguing both coding and non-coding exosomal transcripts. This method provides researchers with strategic options to prepare RNA-Seq libraries and compare RNA-Seq data quantitatively from minimal volumes of fresh and archival human cell-free serum for disease biomarker discovery. (AU)

• Processo FAPESP: 16/06413-3 - Geração de dados epigenômicos de gliomas e de células-tronco com base em plataformas de sequenciamento de larga escala (NGS, next generation sequencing)
• Beneficiário: Marcos Abraão de Souza Fonseca
• Linha de fomento: Bolsas no Brasil - Programa Capacitação - Treinamento Técnico
• Processo FAPESP: 17/08211-1 - Estudo da origem celular do câncer de ovário usando dados genômicos e epigenômicos em larga escala.
• Beneficiário: Marcos Abraão de Souza Fonseca
• Linha de fomento: Bolsas no Brasil - Programa Capacitação - Treinamento Técnico