Patent attributes
The present invention relates to a method for the amplification of mRNA of a sample, comprising the steps of i.) generating cDNA from polyadenylated RNA employing at least one primer hybridizing to said polyadenylated RNA and comprising a 5′ poly(C) or a 5′ poly(G) flank; ii.)(aa) if present, removing non-hybridized, surplus primer(s) and/or surplus dNTPs; ii.)(ab) 3′ tailing of said generated cDNA with a poly(G) tail when in step i.(a) primer(s) comprising a 5′ poly(C) flank was employed or a poly(C) tail when in step i.(a) primer(s) comprising a 5′ poly(G) flank was employed; or ii.)(b) 3′ tailing of said generated cDNA with a poly(G) tail when in step i.(a) primer(s) comprising a 5′ poly(C) flank was employed or a poly(C) tail when in step i.(a) primer(s) comprising a 5′ poly(G) flank was employed using an RNA-ligase, irrespective of the presence or absence of surplus primer(s) and/or surplus dNTPs; and iii.) amplifying the tailed cDNA with a primer hybridizing to the tail(s) generated in step ii(ab) or ii(b). Furthermore, the present invention relates to methods for the preparation of in vitro surrogate(s), for identifying expressed genes in a test sample, for identifying a drug candidate for therapy of a pathological condition and for in vitro detection of a pathological condition employing said method for amplification of mRNA. In addition, the present invention relates to the use of amplified cDNA(s) as obtained by the method of the invention in hybridization, interaction and/or enzymatic arrays.
