Genotyping of Chimerical BCR-ABL1 RNA in Chronic Myeloid Leukemia by Integrated DNA Chip  Jong-Hun Kang, Hyun-Gyung Goh, Sang-Ho Chae, Sung-Yong Kim, Dong-Wook Kim, Chi-Bom Chae  The Journal of Molecular Diagnostics  Volume 14, Issue 5, Pages 487-493 (September 2012) DOI: 10.1016/j.jmoldx.2012.04.003 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Chimerical BCR-ABL1 mRNA and the position of PCR primers and capture oligonucleotide probes. The nucleotide position of the primers and capture probes are based on the sequence of BCR mRNA (GenBank accession no. X02596) and that of ABL1 mRNA (GenBank accession no. X16416). Shown are the exon number of BCR and ABL1 mRNA; B2, B3, and C3 indicate the part of the BCR exon containing the breakpoint. The Journal of Molecular Diagnostics 2012 14, 487-493DOI: (10.1016/j.jmoldx.2012.04.003) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Validation of PCR primers and capture oligonucleotide probes using the plasmids of cloned genotypes of chimerical BCR-ABL1 cDNA. A: Gel electrophoresis of PCR product of plasmids b3a2 (394 and 183 bp, lane 1), b2a2 (319 and 183 bp, lane 2), e1a2 (353 and 183 bp, lane 3), c3a2 (361 bp and 183 bp), and a2 (183 bp). M, size marker. B: The result of hybridization for the same five plasmids. Condition of scanning of slides: photomultiplier tube (PMT) gain 90 and laser power 100. Each plasmid (105 copies) was amplified in a one-step RT-PCR reaction mixture containing all of the primers (Table 1), heat-denatured, and hybridized with the capture probes on the surface of microarray glass slides (as described under Materials and Methods). The final reaction mixture after the completion of hybridization was subjected to gel electrophoresis. The Journal of Molecular Diagnostics 2012 14, 487-493DOI: (10.1016/j.jmoldx.2012.04.003) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Detection of mixed genotypes by the integrated DNA chip. For multiplex analysis, plasmids containing different genotypes of BCR-ABL1 cDNA (105 copies each) were added to one-step RT-PCR reaction mixture, and on-slide PCR and hybridization was performed (Figure 2). A: After on-slide PCR and hybridization, the reaction product was analyzed by gel electrophoresis. Lane 1: b3a2 plus e1a2 plasmid (the PCR product: 394, 353, and 183 bp); lane 2: b2a2 plus e1a2 plasmid (319, 353, and 183 bp); lane 3: b3a2 plus b2a2 plasmid (394, 319, and 183 bp). M, size marker. B: The result of hybridization for the same pairings: b3a2 plus e1a2, b2a2 plus e1a2, and b3a2 plus b2a2 plasmids. When color intensity reaches maximum, the color is red. When the signal is saturated, it is white. The signal of C3-P is black due to absence of c3a2. The Journal of Molecular Diagnostics 2012 14, 487-493DOI: (10.1016/j.jmoldx.2012.04.003) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Increase in signal intensity with integrated reverse transcription, asymmetric PCR, and hybridization. A one-step RT-PCR reaction mixture was assembled with the total cellular RNA containing 104 copies of b3a2 mRNA (from K562 cells). Reverse transcription and symmetric PCR (1; 5 pmol each of B2-F, E1-F, C3-F, and A2-F; 5 pmol of A2-R) was performed and the DNA was purified. The purified DNA was hybridized for 1 hour at 55°C with the capture probes immobilized on a slide in 3× SSC. Reverse transcription and symmetric PCR reaction mixture (2) was assembled as in 1; on-slide reverse transcription, PCR, and hybridization was performed on the surface of the glass slide in a complete reaction mixture (as described under Materials and Methods). Reverse transcription and asymmetric PCR (3; 5 pmol each of B2-F, E1-F, C3-F, and A2-F; 50 pmol of A2-R) was performed and the DNA was purified. The DNA was hybridized with the immobilized capture probe B2-P in 3× SSC. Reverse transcription and asymmetric PCR reaction mixture (4) was assembled as in 3 and on-slide reverse transcription, PCR, heat denaturation, and hybridization were performed in complete RT-PCR reaction mixture. The background signal was subtracted from the signal obtained with the B2-P capture probes (10 spots) on slide. The Journal of Molecular Diagnostics 2012 14, 487-493DOI: (10.1016/j.jmoldx.2012.04.003) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Limit of detection of chimerical BCR-ABL1 mRNA by integrated asymmetric RT-PCR. A: Signal intensity of singleplex and multiplex integrated reverse transcription, PCR, and hybridization versus ratios of b3a2/ABL1 RNA. B: Photograph of capture probe (B2-P and A2-P) spots hybridized with amplified DNA. The relationship between color intensity and relative fluorescence units (RFU) is indicated at the right. The highest intensity was 65,000 RFU and the lowest was 5000 RFU; white represents saturation of color intensity. Different amounts of K562 cellular RNA containing b3a2 mRNA were mixed with HL60 RNA containing a fixed amount of normal ABL1 mRNA (106 copies). The mixed RNA was added to two different sets of asymmetric RT-PCR reaction mixture: singleplex (5 pmol each of B2-F and A2-F; 50 pmol of A2-R) and multiplex (5 pmol each of B2-F, E1-F, C3-F, and A2-F; 50 pmol of A2-R). Integrated on-slide reverse transcription, PCR, and hybridization was performed as described under Materials and Methods. C: Color bar represents the relationship between color intensity and relative fluorescence units (RFU). The highest intensity was 65,000 RFU and the lowest was 50,000 RFU; white represents saturation of color intensity. The Journal of Molecular Diagnostics 2012 14, 487-493DOI: (10.1016/j.jmoldx.2012.04.003) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Identification of the genotypes of chimerical BCR-ABL1 RNA in 15 CML patients. Top: BCR-ABL1 copy no. refers to copy number of chimerical RNA per microgram cellular RNA applied to each microarray chip. Transcript genotype refers to the genotype determined by combined PCR and gel electrophoresis. Integrated process refers to the genotype determined by the integrated DNA chip process. Bottom: Microarray photograph for the same 15 patients and for K562 cell RNA (ie, b3a2 RNA), HL60 cell RNA (ie, normal ABL1 RNA), and water. The RNA from CML patients, which had been previously characterized for genotype by PCR and gel electrophoresis and for copy number of chimerical RNA, was blindly tested for genotype by integrated reverse transcription, asymmetric PCR, and hybridization on a microarray slide (as described under Materials and Methods). The capture oligonucleotide probes were spotted in duplicate on a glass slide, as shown in the template. Fluorescent-labeled oligonucleotide (lambda phage DNA sequence) was spotted in the middle, to serve as position marker. The A2-P capture probe (a2 in the template) serves as normal control for the ABL1 RNA present in normal cells as well as for the A2 exon sequence present in the chimerical RNA (see Figure 1). The Journal of Molecular Diagnostics 2012 14, 487-493DOI: (10.1016/j.jmoldx.2012.04.003) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions