Myeloid Leukemia

208 Cilloni, Gottardi, and Saglio
Fig. 3. Relative quantification using the threshold cycle (Ct) value. The threshold
cycle represents the first cycle of the polymerase chain reaction able to generate a
detectable level of fluorescence. The higher the transcript amount, the smaller the Ct.
corresponding to a logarithmic scale of WT1 and ABL copies. For WT1, we generate
a curve with 100,000, 10,000, 1000, 100, and 10 copies, and for ABL we use
100,000, 10,000 and 1000 copies.
At the end of the run, an Excel final report is provided by the software. The Ct
values and the “quantity” values of standard curve and tested samples are provided.
Ct refers to “threshold cycle,” and it indicates the first cycle of the PCR
reaction in which a detectable level of fluorescence develops and it can be measured
by the instrument (see Fig. 3).
4. Set the threshold to 0.1 and the baseline from cycles 3 to 15 for both WT1 and
ABL genes (see Note 14).
5. Using Excel software, calculate the mean of the Ct and sample quantity from
triplicate wells (see Note 15).
6. Calculate the ratio of WT1 copies/ABL copies and multiply by 10,000 (see Note 16).
7. Samples with an ABL Ct value above 29 must be discarded (see Note 17). The
RQ-PCR assay for WT1 was consistently able to detect 10 plasmid molecules,
and its sensitivity was further established by diluting the K562 cell line into normal
lymphocytes obtained from the peripheral blood of a normal subject that
scored repeatedly negative (Ct ≥ 50) for WT1 expression. Under these experimental
conditions, RQ-PCR was able to detect a positive WT1 signal at a dilution
of one K562 cell in 10 5 normal lymphocytes.
4. Notes
1. Other RNA isolation reagents can also be used (Trizol, Ultraspec, and so on)
with comparable results.