mRNA/cDNA Library Construction Using RNAPolymerase Cycling Reaction

Molecular profiling of single-cell gene expression permits the high-definition investigation of intracellular gene activity and physiological status of the cells under certain special conditions, such as pathogenesis (1 ,2 ), cancer staging (3 ), drug treatment, and developmental processes (4 ). Traditionally, gene transcripts were extracted from lysed cells with pheno-chloroform followed by precipitation, and messenger RNAs (mRNA) were further purified by oligo-(dT)-dextran media (5 ). However, the tedious procedures of extraction, chromatography, and precipitation could not maintain the completeness of a whole mRNA repertoire, resulting in a significant loss of rare RNA (<10 copies/cell) populations. Such loss could be as much as 30% of the original repertoire. The requirement of bulk tissue samples for a better population coverage was another drawback of the pheno-chloroform extraction methods. A minimum of several thousand cells is needed for an acceptable quality of RNA extraction. Because of tissue heterogeneity, these methods usually provided neither reliable nor reproducible results. Unfortunately, it is impossible to collect adequate amounts of pure or homogeneous samples for these methods because of a tremendous difficulty in sample dissection and RNA preservation, especially the preservation of rare mRNA species.