112 Gel & Capillary | RUO Assays Invivoscribe 2019 | 113 Gel & Capillary RUO Assays BCL2/JH t(14;18) Translocation Assay Assay Use The BCL2/JH t(14;18) Translocation Assay is useful for studies involving: •  Monitoring and evaluation of follicular lymphomas and other B-cell lymphomas • Distinguishing lymphoma from benign lymphoid hyperplasia •  Distinguishing follicular lymphoma from other B-cell lymphomas that may have a similar appearance • 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 Five master mixes are included in this assay kit. Two master mixes target BCL2 major break point (MBR) translocations and two target BCL2 minor cluster region (mcr) translocations. An Amplification Control Master Mix is also included to ensure the quality and quantity of sample DNA. Positive and negative controls are also included. This assay can be run either in a standard or nested assay format. Using the standard method, the limit of detection is one cell in one hundred normal cells. The nested method has a limit of detection of one t(14;18) positive cell in a background of ten thousand normal cells. PCR products can be analyzed by standard gel electrophoresis with ethidium bromide staining. A BCL2 translocation is indicated if just one of the 2nd round master mixes (mixes ending in b) generates product(s) within the valid size range. Background The IGH-BCL2 t(14;18)(q32;q21) translocation is found in 80-90% of follicular lymphomas and in 30% of diffuse large cell lymphomas1,2 . The translocation is rarely present in other lymphoproliferative diseases2 . The t(14;18) brings about juxtaposition of BCL2 with the Ig heavy chain joining segment. This leads to a marked increase in expression of BCL2 driven by the Ig heavy chain gene enhancer1 . The BCL2 protein inhibits programmed cell death (apoptosis) leading to cell accumulation2 . The majority of breakpoints on 18q21-22 occur within the major breakpoint region (MBR) in the 3’ untranslated region of exon 3 (60-70% of the cases), and the minor cluster region (mcr) located 3’ to BCL2 exon 3 (20-25% of the cases)1 . Some breakpoints occur at distant loci and will not be identified by this particular test2 . Therefore, a negative result does not completely exclude the presence of a IGH-BCL2 gene rearrangement in the sample. In comparison, Southern blot analysis requires 1-2 weeks, is significantly less sensitive, and has more restrictive Specimen requirements. Specimen Requirements 1.  5 mL of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA; or, 2. Minimum 5 mm cube of tissue; or, 3. 2 μg of genomic DNA; or, 4. Formalin-fixed, paraffin-embedded (FFPE) tissue or slides. Reference 1. MS Lee et al., Science 237: 175-178 (1987). 2. M Crescenzi et al., Proc. Natl. Acad. Sci. USA 85: 4869-4873 (1988). Reagents Ordering Information Catalog # Products Quantity 1-309-0010 BCL2/JH t(14;18) Translocation Assay - Gel Detection 30 reactions 1-309-0030 BCL2/JH t(14;18) Translocation Assay MegaKit - Gel Detection 300 reactions Gel Electrophoresis Detection The data shown was generated using the master mix indicated. Amplified products were run on a 2% agarose/TBE gel. Lane 1 is data generated testing the recommended 100% clonal control DNA; lane 2 is data generated testing a 10% dilution of the recommended clonal control DNA; lane 3 is data generated testing a 1% dilution of the recommended clonal control DNA; lane 4 is data generated testing a 0.1% dilution of the recommended clonal control DNA; and, lane 5 is data generated testing IVS-0000 Polyclonal Control DNA (Cat# 4-092-0010). The IVS-0000 control is used as a negative control for many of our tests. A standard 100 base pair DNA size ladder was run in the lanes flanking the test samples. Figure Legend: Simplified view of the genomic organization of the BCL2 and IGH genes on chromosomes 18 and 14, respectively. Yellow boxes represent the exon regions of the BCL2 gene. Exons of the immunoglobulin heavy chain gene are represented in other colors. The solid black lines represents intron regions, which have been left incompletely spliced to assist in demarcation of the exon segments. MBR and mcr type t(14;18) translocations are shown in the lower portions of the figure with the relative positions of primers and the size of the amplicons generated from the positive control DNAs indicated. Controls Concentration Units in Assay Units in Assay MegaKit IVS-0030 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0031 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0009 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes Master Mixes Target Units in Assay Units in Assay MegaKit BCL2/JH t(14;18) (MBR) Mix 1b Inside BCL2 MBR 1 x 1500 μL tube 10 x 1500 μL tubes BCL2/JH t(14;18) (mcr) Mix 2b Inside BCL2 mcr 1 x 1500 μL tube 10 x 1500 μL tubes BCL2/JH t(14;18) (MBR) Mix 1a Outside BCL2 MBR 1 x 1500 μL tube 10 x 1500 μL tubes BCL2/JH t(14;18) (mcr) Mix 2a Outside BCL2 mcr 1 x 1500 μL tube 10 x 1500 μL tubes Amplification Control HLA-DQa 1 x 1500 μL tube 10 x 1500 μL tubes BCL2/IgH Fusion Gene MBR JH Breakpoints Heavy Chain Gene Chromosome 14q32 BCL2 Gene Chromosome 18q21 BCL2/IgH Fusion Gene mcr JH Breakpoints