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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., (Copyright © 2015 Elsevier B.V. All rights reserved.)