A method of performing quantitative two-step PCR amplification on a sample, comprising amplifying the sample in a first-stage multiplex amplification mixture through a first number of PCR cycles, the amplification mixture comprising a plurality of target primer pairs, each of the target primer pairs configured to amplify one of a plurality of different targets that may be present in the sample in an unknown concentration, and further comprising a plurality of internal quantification standard nucleic acids each of the quantification standards a different sequence and each provided at a different known concentration and at least one quantification standard primer pair, the quantification standard primer pair configured to amplify quantification standard nucleic acids, wherein the quantification standard nucleic acids all have similar amplification efficiencies; dividing the first-stage amplification mixture into a plurality of second-stage individual reactions, a first group of the plurality of second-stage individual reactions each comprising at least one second-stage target primer pair configured to further amplify one of the different targets that may be present in the sample, and a second group of the plurality of second-stage individual reactions each comprising at least one second-stage quantification standard primer pair configured to further amplify one of the quantification standard nucleic acids wherein at least one member of each of the second-stage quantification standard primer pairs is different from that member of each of the other second-stage quantification standard primer pairs; and subjecting the plurality of second-stage individual reactions to a second set of PCR cycles under amplification conditions to generate one or more target amplicons from the plurality of different targets, each target amplicon having an associated target Ct, and a plurality of quantification standard amplicons, each quantification standard amplicon having an associated quantification standard Ct, wherein each target Ct and each quantification standard Ct is determined during the second set of PCR cycles; generating a standard curve from the quantification standard Cts and quantifying each of the one or more targets using the associated target Ct and the standard curve. amplifying the sample in a first-stage multiplex amplification mixture through a first number of PCR cycles, the amplification mixture comprising a plurality of target primer pairs, each of the target primer pairs configured to amplify one of a plurality of different targets that may be present in the sample in an unknown concentration, and further comprising a plurality of internal quantification standard nucleic acids each of the quantification standards a different sequence and each provided at a different known concentration and at least one quantification standard primer pair, the quantification standard primer pair configured to amplify quantification standard nucleic acids, wherein the quantification standard nucleic acids all have similar amplification efficiencies; a plurality of target primer pairs, each of the target primer pairs configured to amplify one of a plurality of different targets that may be present in the sample in an unknown concentration, and further comprising a plurality of internal quantification standard nucleic acids each of the quantification standards a different sequence and each provided at a different known concentration and at least one quantification standard primer pair, the quantification standard primer pair configured to amplify quantification standard nucleic acids, wherein the quantification standard nucleic acids all have similar amplification efficiencies; dividing the first-stage amplification mixture into a plurality of second-stage individual reactions, a first group of the plurality of second-stage individual reactions each comprising at least one second-stage target primer pair configured to further amplify one of the different targets that may be present in the sample, and a second group of the plurality of second-stage individual reactions each comprising at least one second-stage quantification standard primer pair configured to further amplify one of the quantification standard nucleic acids wherein at least one member of each of the second-stage quantification standard primer pairs is different from that member of each of the other second-stage quantification standard primer pairs; and subjecting the plurality of second-stage individual reactions to a second set of PCR cycles under amplification conditions to generate one or more target amplicons from the plurality of different targets, each target amplicon having an associated target Ct, and a plurality of quantification standard amplicons, each quantification standard amplicon having an associated quantification standard Ct, wherein each target Ct and each quantification standard Ct is determined during the second set of PCR cycles;