Myeloid Leukemia

182 Cools, Stover, and Gilliland
Fig. 2. Reverse-transcription polymerase chain reaction (RT-PCR) results. Example
of the RT-PCR results for five hypereosinophilic syndrome cases. Cases 1, 3, 4, and 5
are positive, whereas case 2 is negative. Note that the positive cases show multiple bands
because of alternative splicing and thus the presence of different fusion transcripts.
The FIP1L1-PDGFRA fusion is amplified using the oligonucleotides
FIP1L1-F1 and PDGFRA-R1. At the same time, also set up a PCR reaction for
a control gene (e.g., ZNF384) using primers ZNF384-F and ZNF384-R as a
control for RNA quality and cDNA synthesis. Set up four reactions for each
primer pair, according to Table 1, and perform the PCR according to the program
in Table 2.
3.2.3. Second-Round PCR Reaction
Set up four reactions with the primers FIP1L1-F2 and PDGFRA-R2, according
to Table 1, using 1 µL of the first-round PCR products instead of 1 µL
of cDNA/water. Perform the PCR according to the program in Table 2.
3.2.4. Gel Electrophoresis and Interpretation of the Results
Analyze the PCR products, by loading 5 µL on a 1.5% agarose gel (see Fig. 2).
Expected results are shown in Table 3. Amplification of ZNF384 serves as
a control for RNA quality and cDNA synthesis. If no product is generated after
first-round PCR, then either the RNA of that sample was of bad quality or the
cDNA synthesis failed. The FIP1L1-PDGFRA transcript should be detected
after the first-round PCR in the positive control (EOL-1 cells), as well as after
second-round PCR, and should remain negative after second round of PCR in
the negative and water controls. Only then can the result for the HES/CEL
patient be completely trusted. In most patients, multiple bands are visible on
the gel, due to the presence of splice variants of the FIP1L1-PDGFRA fusion
(as shown in Fig. 2) (5).
3.3. Detection of the FIP1L1-PDGFRA Fusion Gene at the DNA Level
In contrast with the detection of the FIP1L1-PDGFRA fusion transcript at
the RNA level, which can be performed by PCR amplification using primers