Single-Cell mRNA Library Analysis by Northern Blot Hybridization
互联网
485
The debut of RNA-polymerase cycling reaction (RNA-PCR) has promised to provide linear amplification of a reproducible mRNA library from as few as 20 single cells (1 ). By incorporating a RNA promoter element during the synthesis of double-stranded complementary DNA (cDNA) templates, a poly(A+ ) RNA library can be generated and reamplified from the templates in the same conformation and composition as its mRNA origins (Fig. 1 ). Using microarray analysis, the RNA-PCR-derived poly(A+ ) library has been proven to contain above 97% of the original poly(A+ ) RNA population and maintain 88�4% linear correlationship to the populationary ratio of each RNA species (Chapter 12 ). It has also been tested to generate a full-length mRNA library from as few as 20 homologous tissue cells (2-pg mRNAs) for profiling cancer stages in vivo.
Fig. 1. An illustration of using RNA-PCR-derived poly(A+ ) RNA for Northern blot analysis. The mRNA generated in step D can be fractionated on a formaldehydeagarose gel for specific gene detection.
