Patent ID: 8486628

Claim:
A method for normalization of a quantity of a nucleic acid to be quantified in a sample to (i) a total quantity of nucleic acid in the sample; or to (ii) a total quantity of a specific class of nucleic acid in the sample, comprising the steps of (a) providing a sample containing a nucleic acid to be quantified; (b) adding a first nucleic acid probe to the sample under conditions allowing for hybridization of at least a terminal region of said first nucleic acid probe to specific binding sites of said nucleic acid in the sample such that double-stranded nucleic acids are created, wherein said first nucleic acid probe comprises at least one primer binding site, a probe binding site, and an anchor oligonucleotide binding site, and wherein said specific binding sites are a poly-A sequence, a poly-A tail, a poly-T-sequence or a poly-T-tail; (c) adding an anchor oligonucleotide under conditions allowing for hybridization to the anchor oligonucleotide binding site of the first nucleic acid probe so that a 5′ sequence of the anchor oligonucleotide is not hybridized to the first nucleic acid probe; (d) elongating the nucleic acid at its 3′ end, wherein said first nucleic acid probe serves as template until a region is reached where said anchor oligonucleotide is hybridized to the first nucleic acid probe whereon the anchor oligonucleotide serves as a template for further elongation; (e) quantifying the total quantity of nucleic acid in the sample or the total quantity of the specific class of nucleic acid in the sample using a second probe substantially complementary to a region of DNA transcribed from said first nucleic acid probe and said anchor oligonucleotide; (f) quantifying the nucleic acid to be quantified using optionally a third probe whose sequence is substantially identical to a defined region of the nucleic acid to be quantified; and (g) normalizing the quantity of the nucleic acid to be quantified by determining the ratio of the quantity of the nucleic acid to be quantified to the total quantity of nucleic acid in the sample or the total quantity of the specific class of nucleic acid in the sample.