Myeloid Leukemia

FIP1L1-PDGFRA in Hypereosinophilic Syndrome 181
11. Centrifuge at 12,000g for 5 min at 4°C.
12. Remove the ethanol and spin briefly. Remove the rest of the ethanol and air-dry
the pellet for 5 min. Do not dry the pellet for much longer than 5 min, as this may
cause problems with dissolving the RNA.
13. Resuspend the RNA pellet in 10–20 µL RNase-free water, and measure the concentration
(absorption at 260 nm, A 260) and the A 260/A 280 ratio (7).
3.2. Detection of the FIP1L1-PDGFRA Fusion at the RNA Level
The FIP1L1-PDGFRA fusion transcript is a tumor-specific transcript that is
not expressed in normal cells (5). One of the best techniques to detect the presence
of this fusion gene in hypereosinophilic syndrome (HES)/ chronic eosinophilic
leukemia (CEL) patients is the use of reverse-transcription
(RT)-polymerase chain reaction (PCR) with FIP1L1- and PDGFRA-specific
primers (Figs. 1 and 2). This analysis requires the availability of RNA (total
RNA or mRNA) from peripheral blood or bone marrow (see Note 6).
RT-PCR consists of two separate steps: a cDNA synthesis step, followed by
two consecutive PCR steps. In many cases, the FIP1L1-PDGFRA transcript
can be detected after the first PCR reaction, but to increase sensitivity and
specificity, a second (nested) PCR is recommended (see Note 7).
3.2.1. cDNA Synthesis
During random cDNA synthesis, RNA is reverse transcribed to single-strand
cDNA (first-strand cDNA). Use 1–5 µg of total RNA. If you are not experienced
with cDNA synthesis, use a commercial kit that provides all components:
reverse transcriptase, buffer, dNTPs, random hexamer oligonucleotides
(e.g., Superscript First-strand Synthesis System, Invitrogen). During cDNA
synthesis, the RNA and the primers are denatured at 70°C, rapidly cooled down
on ice to keep them denatured, and finally the primers are annealed to the RNA
at 37°C to 42°C. The reverse transcription is usually performed at 37°C to
42°C for 1 h. Follow the recommendations specific for the cDNA synthesis kit.
After cDNA synthesis, the reverse transcriptase enzyme can be inactivated
by a 10-min incubation step at 70°C, but no further purification is required.
3.2.2. First-Round PCR Reaction
For optimal PCR results, it is important to use the correct controls. You will
need a positive control (cDNA in which the FIP1L1-PDGFRA fusion is
present), a negative control (cDNA in which the fusion is not present), and a
water control. A good positive control to use is cDNA obtained from RNA
isolated from the EOL-1 cell line. This cell line expresses the FIP1L1-PDGFRA
fusion gene (8). The negative control can be cDNA generated from RNA from
blood of a healthy individual or from a human cell line that does not express
FIP1L1-PDGFRA.