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.