The multiplexed format of the assay reduces reflex testing by providing quantitation of the three predominant BCR/ABL1 fusion transcripts in a single well. Two types of internal controls are incorporated in the assay, both endogenous (ABL1) and exogenous (BCR/ABL1 Quant Norm). The quantitation of ABL1 provides reporting in BCR/ABL1:ABL1 ratio format, whereas the optional BCR/ABL1 Quant Norm is intended as a process control and allows for normalized absolute quantitation, for example, as copies/mL.
Figure 1: Assay Calibration with Armored RNA® QuantTM (ARQ) Technology.
The copy number of each target and the ratio of BCR/ABL1 to ABL1 copy number are calculated based on 3 calibration curves generated from only 4 multiplex qRT-PCR reactions. In addition, a known fixed copy number of Norm can be spiked in the specimen lysate prior to RNA isolation (optional exogenous internal process control) for monitoring of process efficiency and allowing absolute quantitation of BCR/ABL1, for example, copies/mL of sample.
The kit incorporates a blend of precisely quantified BCR/ABL1, ABL1 and BCR/ABL1 Quant Norm ARQs mixed at different concentrations to generate 3 standard curves (Figure 2). This 3 x 4 Calibrator set is designed to minimize the number of wells used for calibration by using only 4 multiplex qRT-PCR reactions to generate data for 3 standard curves, thereby maximizing the reagents for sample results.
Figure 2: Representative examples of calibration curves†.
Following heat denaturation, the 4 ARQ calibrators were run in triplicate to evaluate repeatability. The resulting Ct values (left) were automatically processed through the ABI 7500 software to build 3 standard curves for BCR/ABL1, ABL1 and BCR/ABL1 Quant Norm (right) covering 5 Logs of copies per RT. R2 were all >0.99.
Assessment of the assay’s analytical sensitivity and linear dynamic range† using serial dilutions of cell line total RNAs showed that a 5 Log decrease of BCR/ABL1 to ABL1 ratio can be reproducibly measured with the assay (Figure 3). In addition, the PCR products are compatible with downstream capillary electrophoresis analysis allowing optional determination of fusion transcript identity by size fractionation, if desired (data not shown). For more information,
Figure 3: Analytical sensitivity†
Samples were prepared by diluting total RNA purified from a cell line expressing b2a2 fusion transcripts in a background of HL-60 RNA and keeping the total RNA input per RT reaction constant at 1,500 ng. About 1,000 copies of Norm transcripts were also spiked into each reaction to mimic specimens purified in the presence of the Norm process control. Samples were tested in duplicate (100, 1 and 0.01%) or in triplicate (0.001 and 0.0001%). The linear dynamic range of the assay was ~6 Logs with 100% detection at 1:100,000 dilution. The same performance was obtained with 500-2,000 ng input of total RNA for all 3 BCR/ABL1 fusion transcripts and all qPCR positive samples were also positive by capillary electrophoresis analysis (data not shown).
BCR/ABL 1 Quant (RUO)* [P/N 46080] 48 Reactions - Kit Configuration.
*Research Use Only. Not intended for use in diagnostic procedures.
