University of Dundee Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes Kennedy,

Background: Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficultly in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics. Citation: Kennedy L, Pauriah M, Godfrey V, Howie J, Dennis H, et al. (2011) Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes. PLoS ONE 6(3): e17625. doi:10.1371/journal.pone. 0017625 Editor: Juan Mata, University of Cambridge, United Kingdom Received November 9, 2010; Accepted February 3, 2011; Published March 22, 2011 Copyright: 2011 Kennedy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Translational Medicine Research Collaboration. GM, CG, DC & GF were employees of Wyeth Research/PGRD and were involved in the study design, data collection and analysis, decision to publish and preparation of the manuscript. Competing Interests: GM, CG, GF & DC are employees of legacy Wyeth Research and latterly Pfizer Global Research & Development (PGRD). Wyeth Research provided funding to the Translational Medicine Research Collaboration (TMRC) of which this study is part. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: g.miele@dundee.ac. uk (GM); c.c. lang@dundee.ac. uk (CL) . These authors contributed equally to this work.