Genomic DNA samples with many amplifications and/or deletions, such as tumor DNA, require users to verify their single-copy reference genes to ensure that the reference genes themselves have not undergone a change in copy number. The advantage of a multi-copy reference assay is demonstrated in the measurement of the relative copy number of RNase P in 2 common cancer cell lines. The absolute average copy numbers of RNase P per normal genome copy amount of DNA were determined in 2 breast cancer cell line (SKBR3 and MCF7) genomic DNAs with the ΔΔCT method, using qBiomarker Multi-Copy Reference Copy Number PCR Assay as the normalization control of DNA input. The absolute copy number of RNase P per normal genome in reference genomic DNA is assumed to be 2. The copy number of RNase P in both SKBR3 and MCF7 is significantly altered, demonstrating that this reference gene would be an unreliable normalizer for these samples.
Tumor cell line DNA (SKBR3) and reference genomic DNA (Promega) were mixed in different ratios (100%, 87.5%, 75%, 50%, 25%, 12.5%, and 0% SKBR3 cells), and the DNA mixes were tested for the gene copy number of GRB7 (a gene that is significantly amplified in SKBR3), using either the MRef assay (A) or the RNase P assay (B) as the reference. The GRB7 copy number in reference DNA is assumed to be 2. The expected GRB7 gene copy numbers in 87.5%, 75%, 50%, 25%, and 12.5% mixing ratio samples are calculated based on the GRB7 gene copy number in 100% SKBR3 gDNA sample, and the mixing ratio between the SKBR3 gDNA and reference genomic DNA.