Quantitative detection of PML-RARalpha fusion transcript by real-time PCR with a single primer pair.
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.
Takenokuchi
Mariko
M
Faculty of Pharmacological Science, Himeji Dokkyo University, Himeji, Hyogo 670-8524, Japan. tmariko@himeji-du.ac.jp
Nakamachi
Yuji
Y
Yoneda
Keiko
K
Joo
Kana
K
Kawano
Seiji
S
Tatsumi
Eiji
E
Saigo
Katsuyasu
K
Kumagai
Shunichi
S
D001854:Bone Marrow Cells / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics* / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000235:genetics* / Q000378:metabolism; D011379:Prognosis; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D015514:Oncogene Proteins, Fusion; D020033:Protein Isoforms; D012334:RNA, Neoplasm; D018168:Receptors, Retinoic Acid; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein