Assay specifications

  Limit of detection   ≤ 10,000 copies/assay wella
  Limit of quantitation   ≤ 20,000 copies/assay wellb
  Linear dynamic range   ≥ 2.5 logsc
  Assay CV   ≤ 15% intra-assay;≤ 20% inter-assay
  Compatible sample types   Cultured cells, bacteria, whole blood, 
  PAXgene blood or dried blood spots, 
  fresh/frozen tissues(human, animal or plant), 
  FFPE samples, purified DNA
  Assay format   96-well plated
  Targets/well   3–33
  Automation compatible
  Yese

Defined as signal greater than background plus three standard deviations of the background.
Defined as the signal just above the lowest signal that obtains an 80–120% spike recovery.
Defined as the assay window that consistently achieves a 80–120% accuracy of fold change.
Contact Affymetrix for information on a 384-well version.
Batch or full automation using standard automation equipment. Contact Technical Support for protocol details and assistance in setting up your automation system.

Multiplex DNA copy number analysis
The QuantiGene™ Plex DNA Assay allows simultaneous detection of multiple copy number alterations in a single well. In the example below, an eight-plex DNA assay was used to quantify Her2 and other genes in SKBR3 breast cancer cells and control cell lines (normal skin fibroblasts and MDA-231 cells) (Figure 1). The QuantiGene Plex DNA Assay showed a ~7-fold amplification of the Her2 gene and two other genes on chromosome 17, and was concordant with that of a bDNA Her2 DNA fluorescent  in situ hybridization (FISH) assay (Figure 2), where a 7-fold amplification of Her2 was detected in SKBR3 cells relative to control HeLa cells.

Figure 1: QuantiGene Plex DNA Assay in SKBR3, normal skin fibroblast, and MDA-231 cells. The ratio of SKBR3 to control copies was ~7. Two other genes on chromosome 17 were amplified and one gene was not. Control genes on chromosomes 1, 5, and 8 in SKBR3 breast cancer cells and control cell lines did not show any DNA copy number amplification.

Same results from any starting material

The QuantiGene Plex DNA Assay offers reproducible and reliable results from a variety of starting materials. In this example, purified genomic DNA as well as lysates from seed and leaves were prepared from the same transgenic lettuce plant. The samples were assessed using the QuantiGene Plex DNA Assay with a panel of 10 targets. After normalization to gene 10, the results indicated the same ratio of gene copy number for each of these genes in all three samples.

Drug metabolism panel – DNA copy number

The QuantiGene Plex DNA Assay offers single-copy number discrimination for accurate classification of copy number variation in drug metabolism studies. The assay can be used as a companion to Affymetrix DMET™ Arrays or to validate array data. In this case, a nine plex assay was used to quantify six target genes, SULT1A1, GSTM1, CYP2D6, CYP2A6, GSTT1, and UGT2B17, normalized using three genes (RPPH1, SNAP25, and PMP22) in 20 Coriell samples (nine Asian, six Yoruba, and five Caucasians) and one ParagonDx sample (Caucasian). The results were analyzed using the background-subtracted average MFI value of target and control genes. The target genes’ MFI value was normalized by the geometric mean of the three control genes’ MFI. The control genes are considered to be N=1. In the data shown, F: female; M: male; J/M: Japanese male; J/F: Japanese female; C/M: Chinese male; and C/F: Chinese female.