Method of quantitative and/or comparative measurement of mRNA expression levels in small biological samples

A method for quantitative and/or comparative assessment of the relative amounts of mRNA transcripts present in a cell or tissue sample, the method comprising the steps of:

• a) carrying out reverse transcription of the mRNA contained in the sample using sequence-modifying primers for several genes in the same reaction to obtain a pool of sequence-modified cDNA molecules, wherein the sequence-modifying primers are oligonucleotides comprising three functional segments:
• i. a 5′-terminal segment comprising a nucleotide sequence, which is complementary to the mRNA sequence as well as the sense strand DNA sequence of a specific gene and contains the complementary nucleotide sequence of the binding sites for the downstream primers used in subsequent amplification reactions,
• ii. a central segment consisting of a nucleotide sequence comprising one or multiple nucleotides, which are non-complementary to the mRNA sequence as well as the sense strand DNA sequence of said gene, and
• iii. a 3′-terminal segment comprising a nucleotide sequence, which is complementary to the mRNA sequence as well as the sense strand DNA sequence of said gene,
• b) removing or inactivating redundant sequence-modifying primers after completion of the reverse transcription,
• c) carrying out individual amplification reactions for each of the analyzed genes, so that in each of the reactions sequence-modified cDNA templates are co-amplified with a reference DNA template, using gene-specific primers to generate measurable amounts of amplification products, wherein the gene-specific primers have binding sites which are located on the same exon,
• d) quantitatively measuring the amounts and determining the relative levels of the amplification products derived from sequence-modified cDNA and reference DNA templates to obtain a gene-specific cDNA over DNA ratio in each of the individual amplification reactions, and
• e) combining the gene-specific cDNA over DNA ratios determined in the individual amplification reactions to generate a sample-specific profile of said ratios that reflects the relative amounts of mRNA transcripts originally present in the sample.