118 Gel & Capillary | RUO Assays Invivoscribe 2019 | 119 Gel & Capillary RUO Assays PML/RARa t(15;17) Translocation Assays Assay Use PML/RARα t(15;17) Translocation Assays are useful for studies involving: • Identification of acute promyelocytic leukemia (APL) • 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 Four master mixes are included in these assay kits. Master mixes are used to amplify complementary DNA (cDNA) produced from specimen(s), as well as positive and negative RNA controls (included). Primers target an internal control transcript (RARA, formerly known as RARα) and the variety of Bcr1, Bcr2, and Bcr3 type transcripts expressed from PML-RARA translocations. The limit of detection for this assay is one PML-RARA positive cell in a background of one hundred thousand normal cells. Amplicon products can be analyzed by differential fluorescence detection using capillary electrophoresis or standard gel electrophoresis. A PML-RARA translocation is indicated if just one of the 2nd round master mixes (Mix 2b or Mix 2c) 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 Acute promyelocytic (M3) leukemia (APL) is a distinct form of acute myeloid leukemia (AML) representing approximately 10% of AMLs. These leukemias often express PML-RARA transcripts from t(15;17) chromosomal translocations that fuse the PML (or MYL) gene on chromosome 15 with the retinoic acid receptor a (RARA) gene on chromosome 171,2,34 . Diagnosis of APL is typically based upon identification of promyelocytes with distinctive morphology plus cytogenetic or molecular detection of these t(15;17) translocations4 . Three PML/RARA translocation patterns have been identified: Type A is the short form (S-form); the breakpoint occurs within the breakpoint cluster region 3 (Bcr3). Type B is the long form (L-form) and the breakpoint occurs within Bcr1. There is a third type B variant or variable form (V-form) whose breakpoint occurs within Bcr2. Identification of the PML-RARA translocation is important in APL because it is correlated with responsiveness to treatment with all- trans retinoic acid (ATRA). Patients incorrectly diagnosed with APL by clinical and morphologic criteria alone are typically unresponsive to treatment with ATRA. APL patients are also at risk for disseminated intravascular coagulation (DIC), which can become more severe during conventional chemotherapy. As a result, there is clearly a need for a rapid, sensitive, and reliable molecular assay that identifies PML- RARA transcripts associated with APL. This Assay uses both non-nested (master mix 1) and nested (master mixes 2a, 2b, and 2c) reverse transcriptase PCR for faster and significantly more sensitive results than cytogenetics or other methods. 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. H De Thé et al., Nature 347: 558-561 (1990). 2. H De Thé et al., Cell 66: 675-684 (1991). 3. A Kakizuka et al., Cell 66: 663-674 (1991). 4. WH Miller et al., Proc. Natl. Acad. Sci. 89: 2694-2698 (1992). Reagents Figure Legend: This figure shows the genomic organization of the PML and RARA genes on chromosomes 15 and 17, respectively. Boxes represent exon regions of the PML (red boxes) and RARA (orange) encoding exons. The solid black line represents intron regions, which were left incompletely spliced to assist in demarcation of the exon segments. Primers are indicated by arrows, and the size of several of the products are indicated below the translocated gene segments. S-form (Bcr3) and L-form (Bcr1) PML-RARA translocations are depicted in the lower portion of the figure. Controls Concentration Units in Assay Units in Assay MegaKit IVS-0020 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 PML/RARα t(15;17) Mix 1 RARA 1 x 1500 μL tube 10 x 1500 μL tubes PML/RARα t(15;17) Mix 2a PML-RARA 1 x 1500 μL tube 10 x 1500 μL tubes PML/RARα t(15;17) Mix 2b S- and L-Forms 1 x 1500 μL tube 10 x 1500 μL tubes PML/RARα t(15;17) Mix 2c L-Form 1 x 1500 μL tube 10 x 1500 μL tubes Ordering Information Catalog # Products Quantity 1-311-0010 PML/RARα t(15;17) Translocation Assay - Gel Detection 30 reactions 1-311-0020 PML/RARα t(15;17) Translocation Assay MegaKit - Gel Detection 300 reactions 1-311-0011 PML/RARα t(15;17) Translocation Assay - ABI Fluorescence Detection 30 reactions 1-311-0021 PML/RARα t(15;17) Translocation Assay MegaKit - ABI Fluorescence Detection 300 reactions Gel Electrophoresis Detection The data shown were 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 the recommended 100% clonal control RNA; lane 2 is data generated testing cDNA synthesized from a 10-4 dilution of the recommended clonal control RNA; and, lane 3 is data generated testing cDNA synthesized from the negative control the negative control IVS-0035 Clonal Control RNA (Cat# 4-089-3070). A standard 100 base pair DNA size ladder was run in the lanes flanking the test samples. Capillary Electrophoresis Detection (ABI) The data shown were generated on an ABI 3100 instrument using the master mix indicated. Panel 1 represents data generated testing cDNA synthesized from the recommended 100% clonal control RNA; panel 2 represents data generated testing cDNA synthesized from a 10-4 dilution of the recommended clonal control RNA; and, panel 3 represents data generated testing cDNA synthesized from the negative control RNA. The positive RNA was diluted into the negative control RNA, IVS-0035 (Cat# 4-089-3070). PML Gene Chromosome 15 RARA Gene Chromosome 17