102 Gel & Capillary | RUO Assays Invivoscribe 2019 | 103 Gel & Capillary RUO Assays T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 Assay Use T-Cell Receptor Gamma Gene Rearrangement Assays are useful for studies involving: •  Identification of clonal T-cell populations highly suggestive of T-cell malignancies • Monitoring and evaluation of disease recurrence • Evaluation of new research and methods in malignancy studies Summary and Explanation of the Test This T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 represents an improved approach to PCR-based clonality testing of lymphoproliferative disorders, as it can detect the vast majority of TCR gamma gene rearrangements with a single multiplex master mix. Importantly, this assay includes, in a single tube, primers for all known groups of TCR gamma variable (Vƴ) region genes and joining (Jƴ) region genes that are involved in rearrangements of T-cell lymphomas. In addition, all reverse primers that target the Jƴ region genes are conjugated with the 6FAM fluorophore. Positive and negative controls, as well as a Specimen Control Size Ladder Master Mix are included. PCR products are analyzed by capillary electrophoresis. Background The human T-cell receptor gamma (TRG, formerly known as TCRG) gene locus on chromosome 7 (7q14) includes 14 Vƴ genes belonging to 4 subgroups, 5 Jƴ segments, and 2 Cƴ genes spread over 200 kilobases. The diversity of this locus has historically complicated PCR-based testing. Our new multiplex PCR assay represents an improvement over existing assays as it can detect the vast majority of TCR gamma gene rearrangements with a single multiplex master mix. This master mix targets all conserved regions within the variable (Vƴ) and joining (Jƴ) region genes that are described in lymphoid malignancies. This is critical for more comprehensive analysis of patient samples, as some T-cell lymphoproliferative disorders involve Vƴ and Jƴ regions that would not be identified with a single Vƴ (1-8) and Jƴ 1/Jƴ 2 primer set. In addition, the polyclonal background that results from the combination of all primers in a single tube produces a more robust and easily interpreted signal with capillary electrophoresis, which aids in the interpretation of small peaks. Competitive amplification of all TRG gene rearrangements allows for identification of a quantitative threshold for a positive result and helps to avoid false positive results. The average size of the TRG gene rearrangement PCR amplicons is 190 nucleotides, with a normal distribution of product sizes between 159 and 207 nucleotides. This protocol should lead to improved product formation from formalin-fixed, paraffin-embedded (FFPE) samples compared to other protocols that yield products of 260 nucleotides or larger. 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 tissue or slides. Reference 1. TC Greiner et al., JMD 4: 137-143 (2002). 2. LC Lawnickie et al., JMD 5: 82-87 (2003). 3. Y Sandberg et al., Leukemia 21: 21 (2007). Reagents Ordering Information Catalog # Products Quantity 1-207-0101 T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 - ABI Fluorescence Detection 33 reactions 1-207-0111 T-Cell Receptor Gamma Gene Rearrangement Assay MegaKit 2.0 - ABI Fluorescence Detection 330 reactions Capillary Electrophoresis Detection (ABI) The data shown was generated using the TCRG-6FAM master mix. Amplified products were run on a capillary electrophoresis ABI 3130xl instrument. Panel 1 displays data generated testing 100% TCRG Positive Control DNA (DNA isolated from a cell line known to have both a Vƴ 9 + Jƴ 1/Jƴ 2 and a Vƴ 10 + Jƴ 1/Jƴ 2 rearrangement); panel 2 displays data generated from the testing of the 5% TCRG Positive Control DNA; and, panel 3 displays data generated from the testing of the polyclonal TCRG Negative Control DNA. Figure Legend: Simple representation of the organization of the T-cell receptor gamma gene on chromosome 7. Black arrows represent the relative positions of primers that target the variable region genes and the downstream joining region gene segments that are involved in rearrangements in T-cell lymphomas. The downstream primers are fluorescently labeled through the incorporation of a 6FAM fluorophore. The amplicon products generated from these rearrangements are detected by capillary electrophoresis. Controls Concentration Units in Assay Units in Assay MegaKit 5% TCRG Positive Control DNA 50 μg/mL 1 x 50 μL tube 5 x 50 μL tube TCRG Negative Control DNA 50 μg/mL 1 x 50 μL tube 5 x 50 μL tube Master Mixes Target Units in Assay Units in Assay MegaKit TCRG - 6FAM Vƴ 1-Vƴ 11 + Jƴ 1/Jƴ 2, Jƴ P, Jƴ P1/Jƴ P2 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 was developed by Invivoscribe. The performance of this assay was reviewed and validated by the EuroClonality/ BIOMED-2 Group. Euroclonality manuscript in preparation: multicenter study with 250 clinical patient specimens.