Myeloid Leukemia

Duplexed QZyme RT-PCR for APL Analysis 139
tial to accurately quantitate PML-RARα transcript levels (see Note 18). This
also provides defined acceptance/rejection criteria from which to minimize
false-negative results (see Table 3). This feature of the duplexed quantitative
assay provides a distinct advantage over qualitative methods.
1. Quantitative data are expressed as an RDC, which equals the ratio of copies of
disease transcripts (expressed as the number of copies of PML-RARα in 50 ng of
patient RNA) to control transcripts (expressed as pg equivalents of BCR in 50 ng
of patient RNA). Calculate RDC with the following equation (see Note 8):
RDC at diagnosis = PML-RARα copy# (per 50 ng of patient RNA)/BCR pg equivalents (per
50 ng of patient RNA)
2. Samples are rejected (no result) or accepted (and subsequently classified)
according to estimates for both the BCR and PML-RARα. Once analyzed, patient
data are classified according to one of four categories: (1) no result, (2) negative (not
detectable), (3) detectable, but not quantifiable, or (4) quantifiable (see Table 3).
3.6. Validation of QZyme Assays
In order to produce data that are quantitative, accurate, specific, and reproducible,
it is important for each laboratory to test the critical parameters of the
assay on the equipment in their laboratory. This will also permit inter-laboratory
standardization of assays for multi-center clinical trials. The critical
parameters to test for real-time quantitative PCR are (1) limit of detection, (2)
limit of quantitation, and (3) reproducibility (intra- and inter-assay variation).
In this subheading, a brief description of how to test these parameters is provided,
and a summary of values for the assays used to validate this method is
presented in Table 4.
3.6.1. Limit of Detection (LOD)
LOD refers to the lowest level of an analyte (e.g., PML-RARa transcript)
that can be detected above background (see Note 19). Background signal is
determined by analyzing 10 replicates of negative controls, i.e., a sample without
the gene transcript of interest. A suitable negative control for PML-RARα
is RNA from Meg-01 (a cell line that does not express the PML-RARα transcript).
As BCR is constitutively expressed, an RNA-containing negative control
does not exist. Water is used as the negative control for BCR. The LOD for
the duplexed QZyme PCR PML-RARα-BCR assay is determined using the following
protocol:
1. Set up duplex QZyme PCR PML-RARα-BCR reactions as outlined under Subheading
3.4.
2. At Subheading 3.4., step 3, add 5 µL of 100 ng/µL Meg-01 total RNA to 10
separate reaction tubes, and 5 µL of water to a further 10 reaction tubes.