116 Gel & Capillary | RUO Assays Invivoscribe 2019 | 117 Gel & Capillary RUO Assays BCR/ABL t(9;22) Translocation Assays Assay Use BCR/ABL t(9;22) Translocation Assays are useful for studies involving: •  Identification of chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) • Monitoring and evaluation of disease recurrence • Detection and assessment of residual disease • Evaluation of new research and methods in malignancy studies Summary and Explanation of the Test The master mixes are included in these assay kits used to amplify complementary DNA (cDNA) produced from specimen(s), and positive and negative RNA controls (included). Primers target an internal control transcript (Abl) and p190-, p210-, and p230-type transcripts expressed from BCR-ABL1 translocations. The limit of detection of this assay is approximately one BCR-ABL1 positive cell in a background of one million normal cells. Amplicon products can be analyzed by capillary electrophoresis or standard gel electrophoresis with ethidium bromide staining. A BCR-ABL1 translocation is indicated if just one of the 2nd round master mixes (Mix 2b, Mix 2c, Mix 3b, Mix 3c, or Mix 3d) generates product(s) of the valid size. Reagents for RNA extraction and reverse transcription are not included. This assay is compatible with all standard RNA extraction and cDNA synthesis methods. This is a qualitative assay and has not been validated for quantitative use. Background The Philadelphia chromosome (Ph1) is a specific chromosomal abnormality that results from reciprocal t(9;22)(q34;q11) chromosome rearrangements that fuse coding regions of the BCR gene, located on chromosome 22, with the ABL receptor independent tyrosine kinase gene on chromosome 91,2 . BCR-ABL1 t(9;22) translocations are present in approximately 95% of chronic myeloid leukemia (CML) patients, 20-50% of adult acute lymphoblastic leukemia (ALL) patients, and 2-10% of pediatric ALL patients2 . Although cytogenetic detection of Ph1 is a hallmark of CML, molecular detection of Ph1-positive cells by nested reverse transcriptase PCR is faster and significantly more sensitive than cytogenetics or other methods. Nearly 50% of cytogenetically Ph1-negative CML cases are positive by reverse transcriptase PCR analysis. This makes reverse transcriptase PCR detection of Ph1-positive cells of value in predicting early disease recurrence and progression for CML and ALL patients that are in apparent clinical remission following bone marrow transplantation3 . Molecular detection provides the opportunity for early intervention and treatment. Thus, molecular testing for chimeric BCR-ABL1 transcripts is utilized both in diagnostic evaluation and post- therapeutic monitoring of CMLs and ALLs. Specimen Requirements 1.  5 mL of peripheral blood, bone marrow biopsy, or bone marrow aspirate anticoagulated with heparin or EDTA; or, 2.  Archived cells frozen in 10% DMSO + 90% Fetal Bovine Serum (FBS). Reference 1. R Kurzrock et al., Ann. Intern. Med. 138: 819-30 (2003). 2. JV Melo. Blood 88: 2375-2384 (1996). 3. JP Radich et al., Blood 85: 2632-2638 (1995). Reagents Figure Legend: This figure shows the genomic organization of the BCR and ABL genes on chromosomes 22 and 9, respectively. Boxes represent exon regions of the ABL (red boxes) and BCR encoding exons (other colors). The solid black line represents intron regions, which have been left incompletely spliced to assist in demarcation of the exon segments. The location of exon regions targeted by labeled and unlabeled primers are indicated by arrows. A p210-type BCR-ABL1 translocation (b3a2 junction) is depicted in the lower portion of the figure along with the control ABL transcript control. Controls Concentration Units in Assay Units in Assay MegaKit IVS-0032 Clonal Control RNA 400 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0011 Clonal Control RNA 400 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0035 Clonal Control RNA 400 μg/mL 1 x 100 μL tube 5 x 100 μL tubes Master Mixes Target Units in Assay Units in Assay MegaKit BCR/ABL t(9;22) Mix 1a Abl 1 x 1500 μL tube 10 x 1500 μL tubes BCR/ABL t(9;22) Mix 2a p190 1 x 1500 μL tube 10 x 1500 μL tubes BCR/ABL t(9;22) Mix 3a p210+230 1 x 1500 μL tube 10 x 1500 μL tubes BCR/ABL t(9;22) Mix 1b Abl 1 x 1500 μL tube 10 x 1500 μL tubes BCR/ABL t(9;22) Mix 2b p190 1 x 1500 μL tube 10 x 1500 μL tubes BCR/ABL t(9;22) Mix 2c p190 1 x 1500 μL tube 10 x 1500 μL tubes BCR/ABL t(9;22) Mix 3b p210+230 1 x 1500 μL tube 10 x 1500 μL tubes BCR/ABL t(9;22) Mix 3c p210+230 1 x 1500 μL tube 10 x 1500 μL tubes BCR/ABL t(9;22) Mix 3d p210+230 1 x 1500 μL tube 10 x 1500 μL tubes Ordering Information Catalog # Products Quantity 1-310-0010 BCR/ABL t(9;22) Translocation Assay - Gel Detection 30 reactions 1-310-0020 BCR/ABL t(9;22) Translocation Assay MegaKit - Gel Detection 300 reactions 1-310-0031 BCR/ABL t(9;22) Translocation Assay - ABI Fluorescence Detection 30 reactions 1-310-0041 BCR/ABL t(9;22) Translocation Assay MegaKit - ABI Fluorescence Detection 300 reactions Gel Electrophoresis Detection The data shown was generated using the master mix indicated. Amplified products were run on a 6% polyacrylamide/TBE gel. Lane 1 is data generated testing cDNA synthesized from an alternative 100% clonal control RNA; lane 2 is data generated testing cDNA synthesized from the recommended 100% clonal control RNA; lane 3 is data generated testing cDNA synthesized from a 10-4 dilution of the recommended clonal control RNA. The positive RNA was diluted into the negative control RNA, IVS-0035. A standard 100 base pair DNA size ladder was run in the lanes flanking the test samples. Capillary Electrophoresis Detection (ABI) The data shown was generated on an ABI 3100 instrument using the master mix indicated. Panel 1 represents data generated testing cDNA synthesized from an alternative 100% control RNA; panel 2 represents data generated testing cDNA synthesized from the recommended 100% clonal control RNA; and, panel 3 represents data generated testing cDNA synthesized from a 10-4 dilution of the recommended clonal control RNA. The positive RNA was diluted into the negative control RNA, IVS- 0035 (Cat# 4-089-3070). BCR Chromosome 22 ABL Chromosome 9 BCR/ABL t(9;22) Translocation p210 Type, b3a2 junction ABL cDNA Control