Methods for differentially quantifying nucleic acids

A method for differentially quantifying nucleic acids in a biological sample using differentially methylated loci, the method comprising:

• providing a solution comprising a methylation-sensitive restriction enzyme;
• providing a biological sample, wherein the biological sample comprises a first plurality of nucleic acids and a second plurality of nucleic acids and wherein each of the first and second pluralities of nucleic acids further comprise a group of methylated nucleic acids and a group of unmethylated nucleic acids;
• forming a digested biological sample by combining the biological sample and the solution, such that the methylation-sensitive restriction enzyme cleaves the unmethylated group of nucleic acids;
• forming a reaction mixture by combining the digested biological sample with:
• a set of amplification oligomers configured to amplify the first and second pluralities of nucleic acids;
• a first group of detection probes configured to anneal to a region of the first plurality of nucleic acids;
• and
• a second group of detection probes configured to anneal to a region of the second plurality of nucleic acids;
• amplifying the reaction mixture to generate a first signal from the first group of detection probes and a second signal from the second group of detection probes;
• determining a ratio comprising a first value derived from the first signal to a second value derived from the second signal;
• and
• identifying the proportion of nucleic acid in the biological sample originating from the first plurality of nucleic acids.