Quantitative multiplex PCR with high dynamic range

A method for measuring amounts of at least two mRNAs in a sample by multiplex PCR, comprising the steps of:

• incubating said at least two mRNAs with a reverse transcriptase under conditions effective to produce a single-stranded cDNA from each mRNA, wherein at least one high-abundance mRNA is present in at least a 100-fold molar excess over at least one low-abundance mRNA, and wherein said mRNAs are present in copy numbers between 102 and 108;
• amplifying said single-stranded cDNAs with a thermostable DNA polymerase at a concentration of at least 0.5 units DNA polymerase/μl and a pair of primers for each single-stranded cDNA under conditions effective to amplify said single-stranded cDNAs in a real-time polymerase chain reaction, whereby the amplification of said low-concentration mRNA is inhibited in the presence of said high-concentration mRNA by not more than 10%, relative to amplification in the absence of said high-concentration mRNA;
• determining the amount of each of said amplified single-stranded cDNAs by real-time measurement of an amount of fluorescent emission, wherein the amount of fluorescent emission provides a quantitative value of the amount of said amplified single-stranded cDNA;
• and
• determining the amount of each of said mRNAs in said sample from the amount of said cDNA derived from said mRNA.