Reports

Polony Multiplex Analysis of Gene Expression (PMAGE) in Mouse Hypertrophic Cardiomyopathy

Jae Bum Kim,^1,2* Gregory J. Porreca,^2* Lei Song,² Steven C. Greenway,² Joshua M. Gorham,² George M. Church,² Christine E. Seidman,^1,2,3 J. G. Seidman²

We describe a sensitive mRNA profiling technology, PMAGE (for "polony multiplex analysis of gene expression"), which detects messenger RNAs (mRNAs) as rare as one transcript per three cells. PMAGE incorporates an improved ligation-based method to sequence 14-nucleotide tags derived from individual mRNA molecules. One sequence tag from each mRNA molecule is amplified onto a separate 1-micrometer bead, denoted as a polymerase colony or polony, and about 5 million polonies are arrayed in a flow cell for parallel sequencing. Using PMAGE, we identified early transcriptional changes that preceded pathological manifestations of hypertrophic cardiomyopathy in mice carrying a disease-causing mutation. PMAGE provided a comprehensive profile of cardiac mRNAs, including low-abundance mRNAs encoding signaling molecules and transcription factors that are likely to participate in disease pathogenesis.

¹ Cardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115, USA.
² Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
³ Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.

^* These authors contributed equally to this work.

To whom correspondence should be addressed. E-mail: seidman{at}genetics.med.harvard.edu (J.G.S.); cseidman{at}genetics.med.harvard.edu (C.E.S.)

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Science 321, 956-960
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Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides.

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PNAS 105, 9145-9150
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M. J. Chaisson and P. A. Pevzner (2008)
Genome Res. 18, 324-330
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Nucleic Acids Res.
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Moderated statistical tests for assessing differences in tag abundance.

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3'-O-modified nucleotides as reversible terminators for pyrosequencing.

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PNAS 104, 16462-16467
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