title: Linear mRNA amplification approach for RNAseq from limited amount of RNA.
abstract: Whole-transcriptome evaluation by next-generation sequencing (NGS) has been widely applied in the investigation of diverse transcriptional scenarios. In many clinical situations, including needle biopsy samples or laser microdissected cells, limited amounts of RNA are usually available for the assessment of the whole transcriptome. Here, we describe an mRNA amplification protocol based on in vitro T7 transcription for transcriptome evaluation by NGS. Initially, we performed RNAseq from two human mammary epithelial cell lines and evaluated several aspects of the transcriptomes generated by linear amplification of Poly (A)(+) mRNA species, including transcript representation, variability and abundance. Our protocol showed to be efficient with respect to full-length transcript coverage and quantitative expression levels. We then evaluated the applicability of using this protocol in a more realistic research scenario, analyzing tumor tissue samples microdissected by laser capture. In order to increase the quantification power of the libraries only the 3' end of transcripts were sequenced. We found highly reproducible RNAseq data among amplified tumor samples, with a median Spearman's correlation of 80%, strongly suggesting that the amplification step and library protocol preparation lead to a consistent transcriptional profile. Altogether, we established a robust protocol for assessing the polyadenylated transcriptome derived from limited amounts of total RNA that is applicable to all NGS platforms.
authors.affiliation: International Research Center, A.C. Camargo Cancer Center, Rua Taguá, 440-1° andar, Liberdade, São Paulo, SP 01508-010, Brazil.
authors.forename: Elisa Napolitano
authors.initials: EN
authors.lastname: Ferreira
authors.affiliation: International Research Center, A.C. Camargo Cancer Center, Rua Taguá, 440-1° andar, Liberdade, São Paulo, SP 01508-010, Brazil.
authors.forename: Gustavo
authors.initials: G
authors.lastname: de Campos Molina
authors.affiliation: Hospital Israelita Albert Einstein, Av. Albert Einstein, 627/701-Bloco A, 2°ss, Morumbi, São Paulo, SP 05652-000, Brazil.
authors.forename: Renato David
authors.initials: RD
authors.lastname: Puga
authors.affiliation: Radiology and Oncology Department, Faculdade de Medicina da Universidade de São Paulo, Avenida Dr° Arnaldo, 251, São Paulo, SP 01246-000, Brazil.
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Nagai
authors.affiliation: International Research Center, A.C. Camargo Cancer Center, Rua Taguá, 440-1° andar, Liberdade, São Paulo, SP 01508-010, Brazil.
authors.forename: Antônio Hugo José Froes Marques
authors.initials: AH
authors.lastname: Campos
authors.affiliation: International Research Center, A.C. Camargo Cancer Center, Rua Taguá, 440-1° andar, Liberdade, São Paulo, SP 01508-010, Brazil.
authors.forename: Gustavo Cardoso
authors.initials: GC
authors.lastname: Guimarães
authors.affiliation: International Research Center, A.C. Camargo Cancer Center, Rua Taguá, 440-1° andar, Liberdade, São Paulo, SP 01508-010, Brazil.
authors.forename: Diana Noronha
authors.initials: DN
authors.lastname: Nunes
authors.affiliation: Division of Hematology/Oncology and Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, 1201 Camino de Salud NE, Albuquerque, NM 87131, USA.
authors.forename: Renata
authors.initials: R
authors.lastname: Pasqualini
authors.affiliation: Division of Hematology/Oncology and Division of Molecular Medicine, Department of Internal Medicine, University of New Mexico School of Medicine, 1201 Camino de Salud NE, Albuquerque, NM 87131, USA.
authors.forename: Wadih
authors.initials: W
authors.lastname: Arap
authors.affiliation: Department of Psychiatry, Faculdade de Medicina da Universidade de São Paulo, Rua Dr Ovidio Pires de Campos, 785, São Paulo, SP 05403-010, Brazil.
authors.forename: Helena
authors.initials: H
authors.lastname: Brentani
authors.affiliation: International Research Center, A.C. Camargo Cancer Center, Rua Taguá, 440-1° andar, Liberdade, São Paulo, SP 01508-010, Brazil; National Institute of Science and Technology in Oncogenomics, São Paulo, Brazil.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Dias-Neto
authors.affiliation: International Research Center, A.C. Camargo Cancer Center, Rua Taguá, 440-1° andar, Liberdade, São Paulo, SP 01508-010, Brazil.
authors.forename: Ricardo R
authors.initials: RR
authors.lastname: Brentani
authors.affiliation: International Research Center, A.C. Camargo Cancer Center, Rua Taguá, 440-1° andar, Liberdade, São Paulo, SP 01508-010, Brazil; National Institute of Science and Technology in Oncogenomics, São Paulo, Brazil. Electronic address: dirce.carraro@cipe.accamargo.org.br.
authors.forename: Dirce Maria
authors.initials: DM
authors.lastname: Carraro
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics; D002460:Cell Line; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D012333:RNA, Messenger
keywords: Laser capture microdissection; Linear mRNA amplification; NGS; RNAseq; T7-in vitro transcription