Abstract
Generating reliable expression profiles from minute cell quantities is critical for scientific discovery and potential clinical applications. Here we present low-quantity digital gene expression (LQ-DGE), an amplification-free approach involving capture of poly(A)+ RNAs from cellular lysates onto poly(dT)-coated sequencing surfaces, followed by on-surface reverse transcription and sequencing. We applied LQ-DGE to profile malignant and nonmalignant mouse and human cells, demonstrating its quantitative power and potential applicability to archival specimens.
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Acknowledgements
We thank K. Kerouac for technical assistance. D.T.T. is supported by the Pancreatic Cancer Action Network–American Association for Cancer Research Research Fellowship, and D.A.H. is supported by the Howard Hughes Medical Institute. This work was supported by US National Human Genome Research Institute grant 1R44HG005279-01 to F.O. and P.M.M.
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F.O. conceived and designed the study; F.O., D.T.T., B.W.B. and S.P. performed the experiments; N.B. provided the cells; F.O., B.S.W. and S.R. performed the computational analyses; F.O., D.T.T., P.M.M. and D.A.H. wrote the paper, which all authors edited.
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F.O. and P.M.M. are employees of Helicos BioSciences Corporation.
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Supplementary Figures 1–15, Supplementary Tables 1–4 and Supplementary Discussion (PDF 2211 kb)
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Ozsolak, F., Ting, D., Wittner, B. et al. Amplification-free digital gene expression profiling from minute cell quantities. Nat Methods 7, 619–621 (2010). https://doi.org/10.1038/nmeth.1480
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DOI: https://doi.org/10.1038/nmeth.1480
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