106 Gel & Capillary | RUO Assays Invivoscribe 2019 | 107 Gel & Capillary RUO Assays TCRG Gene Clonality Assays Assay Use TRG Gene Clonality Assays are useful for studies involving: •  Identification of clonal T-cell populations highly suggestive of T-cell malignancies • Lineage determination of leukemias and lymphomas • 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 This assay tests all variable (Vƴ ) regions 1-11 of the TRG (formerly known as TCRG) gene.. Master mix tubes A and B target conserved regions within the variable (Vƴ ) and joining (Jƴ ) regions that flank the unique, hypervariable, antigen-binding, complementarity determining region 3 (CDR3). Tube A contains two Vƴ primers and two Jƴ primers. Tube B contains two Vƴ primers and two Jƴ primers. Positive and negative controls, as well as a Specimen Control Size Ladder Master Mix are included. PCR products can be analyzed by capillary electrophoresis or heteroduplex analysis. Clonality is indicated if any one of the master mixes generates clonal products. Background The T-cell receptor gamma (TRG, formerly known as TCRG) chain locus spans 128 kb on chromosome 7 (7p14). Rearrangement of the variable (Vƴ ) and joining (Jƴ ) genetic segments of the TRG locus result in Vƴ-Jƴ products of unique length and sequence. The TRG locus does not contain diversity (Dƴ) segments2 . TRG is a preferential target for clonality analyses since it is rearranged in greater than 90% of T-ALL, T-large granular lymphocyte (LGL), and T-PLL, in 50-75% of peripheral T-NHL and mycosis fungoides, but not NK cell proliferations. It is also rearranged in a major part (60%) of B-lineage ALLs and in a much smaller part of B NHLs. In addition, the TRG gene contains a limited number of Vƴ and Jƴ segments such that the amplification of all major Vƴ-Jƴ combinations is possible with four Vƴ and two Jƴ primers. This standardized multiplex PCR assay detects the vast majority of clonal TRG gene rearrangements using only two multiplex master mixes1 . The potential risk of false-positive results, due to overinterpretation of minor clonal peaks, can be minimized by the combined use of heteroduplex analysis and differential fluorescence detection, and by interpreting results within their clinical context1,2 . The detection rate of clonal TRG gene rearrangements using this assay is exceptionally high1 . The performance characteristics of this assay have been independently determined by the EuroClonality/ BIOMED-2 Group. This consortium of 47 molecular diagnostic laboratories tested hundreds of clinical samples, taking into account their lymphoproliferative status as defined by the WHO classifications2 . 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. K Beldjord et al., Leukemia 17: 2289-2292 (2003). 2. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003). Reagents Ordering Information Catalog # Products Quantity 1-207-0020 TRG Gene Clonality Assay - Gel Detection 33 reactions 1-207-0040 TRG Gene Clonality Assay MegaKit - Gel Detection 330 reactions 1-207-0021 TRG Gene Clonality Assay - ABI Fluorescence Detection 33 reactions 1-207-0041 TRG Gene Clonality Assay MegaKit - ABI Fluorescence Detection 330 reactions Gel Electrophoresis Detection The data shown was generated using the master mix indicated. Amplified products were heteroduplexed and run on a 6% non-denaturing polyacrylamide/TBE gel. Lane 1 is data generated testing an alternative 100% clonal control DNA; lane 2 is data generated testing the recommended 100% clonal control DNA; lane 3 is data generated testing a 10% dilution of the recommended clonal control DNA; and, lane 4 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. Capillary Electrophoresis Detection (ABI) The data shown was generated using the master mix indicated. Amplified products were run on an ABI 3100 instrument. Panel 1 displays data generated testing an alternative 100% clonal control DNA; panel 2 displays data generated testing the recommended 100% clonal control DNA; panel 3 displays data generated testing a 10% dilution of the recommended clonal control DNA; and, panel 4 displays data generated testing the IVS-0000 Polyclonal Control DNA (Cat# 4-092-0010). Our IVS-0000 DNA is often tested to provide information regarding valid size ranges for each master mix. Figure Legend: Depicted is a simple representation of the organization of the T-cell receptor gamma chain gene on chromosome 7. Black arrows represent the relative positions of primers that target the variable (Vƴ) regions, and the downstream joining (Jƴ) gene segments. The amplicon products generated from each of these regions can be differentially detected when fluorescent primer sets are used with capillary electrophoresis instruments that employ differential fluorescence detection. Controls Concentration Units in Assay Units in Assay MegaKit IVS-0009 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0021 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0000 Polyclonal 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 TCRG Tube A Vƴ 1-8 + Vƴ 10 + Jƴ 1 x 1500 μL tube 10 x 1500 μL tubes TCRG Tube B Vƴ 9 + Vƴ 11 + Jƴ 1 x 1500 μL tube 10 x 1500 μL tubes Specimen Control Size Ladder Multiple Genes 1 x 1500 μL tube 10 x 1500 μL tubes This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.