Quantitative detection of PML‐RARα fusion transcript by real‐time PCR with a single primer pair

Abstract

Quantitative detection of minimal residual disease has prognostic value for some leukemias. Acute promyelocytic leukemia (APL) is characterized by the specific PML-RARalpha fusion gene from t(15;17). Added to three PML-RARalpha isoforms, alternative spliced forms of PML exons give rise to multiple isoforms even within a single patient. To date, multiple primer pairs for the detection of the various PML-RARalpha transcripts have been designed, potentially generating some nonspecific amplification products. Here, we established a real-time quantitative PCR (RQ-PCR) strategy with a single primer pair using LightCycler (sp-RQ-PCR), which could simultaneously detect three isoforms with equal specificity and sensitivity as well as alternative spliced forms. Results obtained with sp-RQ-PCR for 39 samples from 15 APL patients and 31 non-APL samples were compared with those with TaqMan assay with three primer pairs. In two of the APL samples, PML-RARalpha was detected in the TM, but not in the sp-RQ-PCR or nested PCR. Furthermore, the sp-RQ-PCR showed no positive results for the 31 non-APL samples, whereas the TM identified 13% (4/31) as positive. Electrophoresis detected some artifacts in the TM, which do not correspond to PML-RARalpha. We conclude that our sp-RQ-PCR is specific enough to identify various forms of PML-RARalpha and yields no false-positive results.

Development and Validation of a 3-Plex RT-qPCR Assay for the Simultaneous Detection and Quantitation of the Three PML-RARa Fusion Transcripts in Acute Promyelocytic Leukemia

“…In any case, we emphasized the strict manipulation rules according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [ 33 ]. The developed assay showed a sensitivity of 10 copies plasmid or 10 -4 RNA cell line dilution for PML-RARa, and this sensitivity is comparable to other studies [ 17 , 18 , 31 ]. Moreover the CVs of inter- and intra-assays were less than 3% for all the three transcripts, indicating that this assay is sensitive and reliable for PML-RARa detection.…”

“…Some studies reported that HybProbe-based RT-qPCR assays allow the detection of the PML-RARa fusion transcripts [ 30 , 31 ]. TaqMan-based RT-qPCR is the most appropriate method in clinical diagnostic laboratories considering cost, turnaround time, and assay performance [ 32 ].…”

Expression of the novel NUP98/PSIP1 fusion transcripts in myelodysplastic syndrome with t(9;11)(p22;p15)

“…Designation of primers for NUP98/PSIP1 fusion transcripts and RT‐PCR using AmpliTaq GOLD 360 Master Mix (Applied Biosystems, Foster City, CA, USA) were performed as described previously (6, 11).…”

“…Spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) We used SkyPaint kit (Applied Spectral Imaging, Migdal Ha'Emek, Israel) and NUP98 ⁄ D11Z1 dual color probe (SRL Inc., Tokyo, Japan) for SKY and FISH, respectively. Mix (Applied Biosystems, Foster City, CA, USA) were performed as described previously (6,11). RQ-PCR was carried out with N988F primer and PSIP1ex8R primer from exon 8 (5¢-TGGGTCTGCC TCTTGGTTTTGGA-3¢), Power SYBR Green PCR Master Mix, and 7900HT Fast Real-time PCR system (Applied Biosystems).…”

FLT3/ITD Associated with an Immature Immunophenotype in PML-RARα Leukemia

“…The PML-RARα transcript for each patient was also measured by the real-time quantitative PCR (RQ-PCR) method that we recently established. 22 PCR for TdT detection was performed as previously described. 23 …”