Kim Jae Bum, Porreca Gregory J, Song Lei, Greenway Steven C, Gorham Joshua M, Church George M, Seidman Christine E, Seidman J G
Cardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115, USA.
Science. 2007 Jun 8;316(5830):1481-4. doi: 10.1126/science.1137325.
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.
我们描述了一种灵敏的mRNA分析技术,即PMAGE(“基因表达的聚合酶克隆多重分析”),它能检测到每三个细胞中仅有一个转录本那么稀少的信使RNA(mRNA)。PMAGE采用了一种改进的基于连接的方法,对源自单个mRNA分子的14个核苷酸标签进行测序。每个mRNA分子的一个序列标签被扩增到一个单独的1微米珠子上,即聚合酶克隆或克隆集落,约500万个克隆集落在流动槽中排列以便进行平行测序。利用PMAGE,我们在携带致病突变的小鼠中识别出肥厚型心肌病病理表现之前的早期转录变化。PMAGE提供了心脏mRNA的全面图谱,包括编码可能参与疾病发病机制的信号分子和转录因子的低丰度mRNA。