BCR/ABL1 Quant*
BCR/ABL1 Quant (RUO)* is a research tool utilizing multiplex real-time quantitative
RT-PCR to provide simultaneous detection and quantification of BCR/ABL1 fusion transcripts
(b2a2, b3a2, and e1a2), ABL1 (an endogenous control), and BCR/ABL1 Quant Norm (an
exogenous control) in a single reaction. The kit is based on TaqMan®
technology and is compatible with ABI 7500 Real-Time Systems or equivalent.
BCR/ABL1 Quant is a state of the art assay providing broad target coverage and dynamic
range with internal and external assay calibration powered by Asuragen’s Armored
RNA® QuantTM (ARQ) Technology (Figure 1).
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. Click to enlarge
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, please click here.
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
For ordering information, please contact orders@asuragen.com.
For more information about capillary electrophoresis for differentiation of the
fusion transcripts via size fractionation,
please click here.
*Research Use Only. Not intended for use in diagnostic procedures.
†Preliminary research data, the performance characteristics of
this assay are not yet established.
If you wish to learn more about quantitative BCR-ABL testing and the International Scale (IS) you can visit our educational web site www.whereareyouontheis.com