GENE PROFILING OF SINGLE OR MULTIPLE CELLS

摘要

An mRNA is amplified by (a) binding a first primer to a target mRNA, the first primer comprising, in the 5 to 3 direction, a first known segment and an oligo T segment; (b) transcribing a cDNA from said target mRNA by elongation of said first primer with reverse transcriptase; and then (c) linking a second known segment to the 3 terminus of said cDNA. In a preferred embodiment the step of transcribing a cDNA from said target mRNA is carried out so that at least one additional C residue is produced on the 3 terminus of said cDNA, and the said step of linking a second known segment to the 3 terminus of said cDNA is carried out by: (i) binding a second bridge primer to said cDNA, said second primer comprising, in the 5 to 3 direction, a second known segment and at least one G residue, said second primer having an inactivated G residue on the 3 terminus thereof; and then (ii) further transcribing said cDNA from second bridge primer by elongation of said at least one additional C residue with reverse transcriptase so that a cDNA is produced having said first known segment on the 5 terminus thereof and said second known segment on the 3 terminus thereof. The method can be used to amplify a plurality of mRNAs together, even though a small sample of mRNAs is available such as single or multiple cell which obtained by laser capture microdissection from tissue or organs, and the product of the amplification then used for gene family analysis or microarray expression analysis.