SuperTCRExpress TM Human TCR Vβ Repertoire CDR3 Diversity Determination (Spectratyping) and Quantitative Analysis Kit

Transcription

1 SuperTCRExpress TM Human TCR Vβ Repertoire CDR3 Diversity Determination (Spectratyping) and Quantitative Analysis Kit Cat. No. H0521 Size: 2 sets (22 Vβ families/each, with enzymes) H0522 Size: 4 sets (22 Vβ families/each, with enzymes) H0536 Size: 8 sets (22 Vβ families/each, with enzymes) Upon receipt of kit, take out enzymes and store at -20 C. Store balance of kit at 4 C. Description The SuperTCRExpress TM Human T Cell Receptor (TCR) Vβ Repertoire CDR3 Diversity Determination (Spectratyping) and Quantitative Analysis System is designed for the convenient, sensitive, reproducible, and quantitative determination of changes in the CDR3 diversity (clonality) of the TCR Vβ repertoires in human T lymphocytes. The system consists of four major components: RNA purification, one step PCR and then nested PCR amplifications without initial cdna synthesis, CDR3 diversity determination, and the quantitative analysis of the CDR3 diversity with proprietary software. The uniquely designed primers and essential reagents have been pre-loaded in a 24-well PCR plate with chimney wells, which is more effective than flat-top plates in preventing contamination. For high resolution spectratyping and easy to perform molecular cloning, seven non-template nucleotides are added to the 5 end of the primers and all of the Vβ primers are labeled with 6-FAM fluorescence. Proprietary PCR reaction buffers and conditions play a critical role, resulting in highly sensitive and specific PCR amplifications. The analysis system is a DNA fragment - based assay. The amplified nested PCR products labeled with 6-FAM can be analyzed for size on many kinds of DNA sequencers using the manufacturer s fragment sizing software. For statistical and quantitative analysis of the CDR3 diversity, our system provides powerful proprietary software. There are two panels (control and testing panel) in the software. The Control panel is used to establish a standard distribution for each CDR3 length of each Vβ family. The extent of changes of CDR3 diversity is defined as D (distance from mean value). D > 3 SD (standard deviations) of each fragment length of each family indicates significant changes in the Vβ family (see the software instruction). The system can detect 22 individual Vβ gene families (from Vβ1 to 24, with the exception of the non-functional Vβ 10 and 19) in human T lymphocytes and their subsets, such as CD4, CD8 and CD45RA and CD45RO T cell subsets of the CD4 and CD8 populations. The kit can be used to determine quantitatively changes in CDR3 diversity (clonality), which are induced by viral, tumor, or selfimmune antigen response and document efficacy of vaccines or immunologic therapy. The kits also can quantitatively indicate T cell immune reconstitution following bone marrow transplantation or combination therapy (HAART) in HIV infected patients.

2 Contents Purpose Name H0521 H0522 H0523 For RNA purification For PCR amplification For CDR3 diversity analysis Buffer A 2 ml 4 ml 8 ml Buffer B 2 ml 4 ml 8 ml Buffer C 2 ml 4 ml 8 ml RNase free water 1 ml 2 ml 4 ml Spin Columns Collection tubes SupHQ plate I* SupHQ plate II** PCR reaction buffer I 200 µl 400 µl 800 µl PCR reaction buffer II 200 µl 400 µl 800 µl Molecular grade water 4 ml 8 ml 16 ml Enzyme mix 14 µl 28 µl 56 µl Taq DNA polymerase 12 µl 24 µl 48 µl Proprietary software * SupHQ plate I is designed to perform primary PCR amplification of 22 Vβ gene families. ** In SupHQ plate II, the 22 nested Vβ primers are labeled with 6-FAM. Protocol RNA isolation Combined cdna synthesis and PCR amplification CDR3 diversity determination (spectratyping) CDR3 diversity analysis using proprietary software

3 A) RNA isolation Open the foil-based self sealing bag labeled RNA isolation 1) Add 350 µl Buffer A to the T cell pellets (1 5 x 10 6 ). Vortex to mix. 2) Add 350 µl of 70% ethanol to the lysate, mix again. 3) Apply all of the sample to the spin column, centrifuge for 15 s at rpm in bench 4) Add 700 µl of Buffer B to the spin column, centrifuge for 15 s at rpm in bench 5) Add 500 µl of Buffer C to the spin column, centrifuge for 15 s at rpm in bench 6) Repeat step 5 7) Spin the column for 1 min to ensure removal of all the Buffer C. 8) To elute, transfer the column to a collection tube (supplied). Add 35 µl RNase-free water directly onto the central of the column, centrifuge for 1 min at rpm in bench top centrifuge. Repeat the elution step with a second volume of RNase-free water. 9) Keep the resulting RNA at -20 o C until use. B) PCR amplifications 1. One step PCR amplification in SupHQ plate I with 24 wells Open the foil-based self sealing bag labeled SupHQ plate I (note the plate and the plate user manual card) For sample tubes (22 Vβ families) While keeping the SupHQ plate I on ice, add the following to each well or make a master mix of the following reagents for each family desired, and aliquot x µl into each sample well of the plate: RNA 3.0 µl PCR reaction buffer I 1.0 µl Enzyme mix 0.3 µl RNase-free water 5.7 µl For control tubes (positive and negative) Add the following reagents to the appropriate tubes: PCR reaction buffer I 1.0 µl Enzyme mix 0.3 µl Molecular grade water 8.7 µl Cover the wells with the provided strip caps and gently vortex the plate to mix. Centrifuge briefly to collect all the reagents at the bottom of the wells or gently tap the plate on a solid surface. Perform PCR amplification in a thermocycler using the following parameters:

4 PCR amplification conditions: 1) 42 o C for 50 min for reverse transcription 2) 94 o C for 3 min 3) 94 o C for 30 sec 4) 55 o C for 30 sec 5) 72 o C for 45 sec Repeat steps 3 to 5 for 35 cycles 6) 72 o C for 5 min Store the PCR products at -20 o C. 2. Nested PCR amplification in SupHQ plate II with 24 wells Open the foil-based self sealing bag labeled SupHQ plate II (note the plate and the plate user manual card) While keeping the SupHQ plate II on ice, add the following to each well or make a master mix of the following reagents for each family desired plus the controls, and aliquot x µl into each sample and control well of the plate: PCR reaction buffer II 2.5 µl Taq DNA polymerase (5U/µl) 0.25 µl Molecular grade water µl Transfer carefully 1 µl of the primary PCR Products with multichannel pipette from each sample and control well in SupHQ plate I to the corresponding wells (samples and controls) in the SupHQ plate II. Cover the wells with the provided strip caps and gently vortex the plate to mix. Centrifuge briefly to collect all the reagents at the bottom of the wells or gently tap the plate on a solid surface. Perform PCR amplification in a thermocycler using the following parameters: PCR amplification conditions: 1) Initial denaturation at 95 o C for 3 min, then 2) 95 o C for 30 sec 3) 55 o C for 30 sec 4) 72 o C for 30 sec Repeat steps 2 to 4, for 25 cycles 5) 72 o C for 10 min Store PCR products at -20 o C.

5 C) CDR3 diversity determination (spectratyping) 1. Checking presence of PCR products Mix 3 µl of the nested PCR product with 0.5 µl of 6 x DNA loading dye, then electrophorese in a 2% agarose (molecular biology grade, not provided by kit) gel. Successful PCR amplifications should display a defined DNA band on the agarose gel from positive control and each sample (each Vβ family) and no PCR product from the negative control well. PCR product sizes of 22 Vβ gene families should be between 120 to 240 bp. 2. TCR CDR3 diversity determination (spectratyping) Samples for analysis on a DNA sequencer are prepared based on the manufacturer s suggestions and using Rox 400 as the size standard. The size of the individual PCR products will be determined by the sequencer s software and should be output as an Excel file for use in the kit s proprietary software. D) CDR3 diversity analysis Quantitative determination of CDR3 diversity of 22 Vβ families can be performed using the proprietary statistical quantitative software package provided in the kit. There are two panels (control and testing panel) in the software. The Control panel is used to establish a standard distribution for size range of each Vβ family. An average distribution of each CDR3 length within the determined T cell population is based on six to ten control samples, which are subjected to the same PCR amplification and spectratyping conditions as the final test samples. The extent of the change in the CDR3 diversity is defined as D (distance from mean value). D > 3 SD (standard deviation) in the fragment length of each family indicates that there are significant changes in the Vβ family (see the software instruction). Optional: CDR3 region sequencing A TA cloning kit (not supplied with the kit) is recommended. Purify 20 µl of PCR products from the Vβ family PCR of interest using a PCR purification kit. Following the manufacturers directions, ligate and transform the DNA fragment into the cloning vector and cells for subsequent sequencing. The M13 forward or reverse primer is recommended for sequencing. Troubleshooting guide Problem Likely Cause Suggestions No signal in samples and positive Sample and control cdna were Be sure the enzyme mix or control not amplified or no cdna was Taq polymerase were No signal in very few Vβ families but signal appears in most Vβ families and the positive control Signal appears in negative control well Poor resolution of CDR3 patterns from in gel visualization synthesized cdna concentration is too low or there are deletions of the individual Vβ family from progressing cells Contamination during PCR preparation PCR product concentration is too high or too low added Increase cdna concentration by increasing the starting number of T cells Decontaminate all equipment, including pipettes. Use filtered pipette tips. Adjust ratio of PCR products with distilled water before running on gel