Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes

Abstract

The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called "the Eprobe leukemia assay," for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.

Fig 1. Schematic illustration of polymerase chain reaction (PCR) Eprobe melting curve analysis.

(A) Eprobe mediated reverse transcription quantitative PCR (RT-qPCR). Fusion transcripts were amplified in a single-tube setting by using standard PCR primers with Eprobe. (B) Melting curve analysis. Eprobe binds to complementary DNA with higher affinity than normal oligonucleotides by exploiting cationic dye moieties, leading to a competitive effect in primer annealing and extension. Accordingly, melting curve analysis can be used to confirm that a single amplicon has been detected by the Eprobe.

Fig 2. Correlation of major and minor BCR-ABL1 mRNA expression levels in double-positive chronic myeloid leukemia (CML) cases.

Major and minor BCR-ABL1 mRNA expression levels were compared in the minor BCR-ABL1 co-expressed specimens obtained from major BCR-ABL1-positive CML cases (11/19, 58%) by TaqMan RT-qPCR.

Fig 3. Abnormal amplification curves in primary samples with low transcript levels of minor BCR-ABL1.

(A) The abnormally low degree of PCR amplification in primary samples with low-level minor BCR-ABL1 transcripts (pt #20 and #21, S1 Table) detected by Eprobe mediated RT-qPCR (performed with the old primer sets indicated in Table 2). (B) Normal amplification curve lines in synthetic minor BCR-ABL1 standard RNA detected by Eprobe mediated RT-qPCR with the old primer set (Table 2).

Fig 4. Non-specific amplification of co-existing RNA interferes the detection of minimal expression of minor BCR-ABL1.

(A) Eprobe fluorescent signals of the Eprobe RT-qPCR products detected by agarose gel (1.5%) electrophoresis. PC depicts positive control that contains synthetic minor BCR-ABL1 transcripts (1.0 × 10⁷ copies/reaction); #20 and #21, primary samples with low expression of minor BCR-ABL1 transcripts, respectively. (B) Same series of samples as shown in panel (A) stained with ethidium bromide. (C) Amplification curve lines of serially diluted synthetic minor BCR-ABL1-spiked-in samples. *Copy numbers of spiked-in synthetic minor BCR-ABL1/reaction in total RNA of HL60 cells (1μg) has been shown.

Fig 5. New primer candidates of minor BCR-ABL1 for Eprobe mediated RT-qPCR.

Fig 6. Amplification curves of the Eprobe mediated RT-qPCR with various combinations of the primer sets using synthetic RNAs and patient samples.

(A) Eprobe RT-qPCR amplification curve lines of minor BCR-ABL1 using various sets of primers. Samples of known concentration were used: (1) synthetic minor BCR-ABL1 RNA (63 copies/reaction)-spiked-in HL60 total RNA; (2) 1,250 copies/reaction spiked-in; (3) 2.5×10⁴ copies/reaction spiked-in; (4) 5.0×10⁵ copies/reaction spiked-in; (5)1.0×10⁷ copies/reaction spiked-in. Red lines depict synthetic minor BCR-ABL1 RNA with HL60 total RNA; green lines, synthetic minor BCR-ABL1 RNA without HL60 total RNA. (B) Ampification curves in major and minor BCR-ABL1 dual-positive CML primary samples.