SuperTCR ExpressTM Mouse T Cell Receptor (TCR) V ββββ Repertoire Clonality Detecting Kit

Cat. No. M0543 Size: 2 sets (22 V ββββ families/each, w/o enzymes)

M0544 Size: 4 sets (22 V ββββ families/each, w/o enzymes)

M0562 Size: 8 sets (22 V ββββ families/each, w/o enzymes)

Store at 4°C

Description The SuperTCRExpressTM Mouse T Cell Receptor (TCR) Vββββ Repertoire Clonality Detecting System is designed for the quick, sensitive, and reproducible detection and analysis of clonality of the TCR Vβ repertoires in CDR3 region in mouse T cell population. The assay system consists of three major components: RNA purification, one step and then nested PCR amplifications without initial cDNA synthesis step, and finally the visualization of the clonality assay. The system provides columns and reagents for total RNA purification. The uniquely designed primers including Vβ, Dβ and Jβ gene primers and essential reagents have been pre-loated in a 24- well PCR plate with chimney wells. The process of a one step PCR amplification makes the reactions easy to handle and reduces the potential for contamination. Proprietary PCR reaction buffer and conditions play a critical role resulting in highly sensitive and specific PCR amplifications. The clonality detection is multiple PCR amplification and gel - based assay (using a standard horizontal gel tray) and does not require a more costly DNA sequencer for reliable results. The high resolution (HR) gel provided in the kit is essential for the clonality assay and can be stained with ethidium bromide to visualize the bands of data. The resolution of a 4% gel approaches that of an 8% polyacrylamide gel. Therefore, the amplified Vβ gene of monoclonal expanded T cells or of oligoclonal expanded T cells displays either a single band or multiple defined bands, while the amplified genes of polyclonal T cells show a smear with no defined bands. The kit provides a special 10 bp DNA size standard to serve as a molecular marker in the assay. Moreover, bands of interest can directly be cut from the gel for sequencing of the CDR3 region. The SuperTCRExpressTM Mouse T Cell Receptor (TCR) Vβ Repertoire Clonality Detecting System can identify 22 individual Vβ gene families (from Vβ1 to 20, Vβ8 of them has three subfamilies, Vβ8.1, Vβ8.2, and Vβ8.3) in mouse 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 detect TCR clonalities that are induced by of viral, tumor, or self- immune antigen induced TCR disruptions and the efficacy of vaccines or immunologic therapy in mouse model. The assay can be completed in just a few hours.

Contents

Purpose Name M0543 M0544 M0562 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 2 4 8

For RNA purification

Collection tubes 4 6 10 SupMC plate I* 2 4 8 SupMC plate II* 2 4 8

PCR reaction buffer I** 200 µl 400 µl 800 µl PCR reaction buffer II** 200 µl 400 µl 800 µl

For PCR

amplification Molecular grade water 4 ml 8 ml 16 ml

High resolution (HR) gel 8 g 16 g 32 g 6 x DNA loading dye 100 µl 200 µl 400 µl

For Clonality Assay

10 bp DNA size standard 20 µl 40 µl 80 µl

* SupMC plate I and SupMC plate II are designed to amplify 22 Vβ gene families (Vβ1 to Vβ20, thereof Vβ8 with three sub families, Vβ 8.1, Vβ 8.2 and Vβ 8.3).

** Enzymes are not provided in this kit. User provi des all enzymes.

Protocol RNA isolation

↓ Combined cDNA synthesis and PCR amplifications

↓ TCR Clonality assay

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 106). 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 ≥10000 rpm in bench

top centrifuge. Discard the flow through. 4) Add 700 µl of Buffer B to the spin column, centrifuge for 15 s at ≥10000 rpm in bench

top centrifuge. Discard the flow through. 5) Add 500 µl of Buffer C to the spin column, centrifuge for 15 s at ≥10000 rpm in bench

top centrifuge. Discard the flow through. 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 ≥10000 rpm in bench top centrifuge. Repeat the elution step with a second volume of RNase-free water.

9) Keep the resulting RNA at -20oC until use.

B) Combined cDNA synthesis and PCR amplifications

1. One step PCR amplification in SupMC plate I with 24 tubes •••• Open the foil-based self sealing bag labeled “SupMC plate I” (note the plate and the plate

user manual card) For sample tubes (22 Vββββ families) While keeping the SupMC 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 MMLV reverse transcriptase (200U/µl) 0.2 µl (provided by user) Taq DNA polymerase (5U/µl) 0.1 µl (provided by user) 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 MMLV reverse transcriptase (200U/µl) 0.2 µl (provided by user) Taq DNA polymerase (5U/µl) 0.1 µl (provided by user) 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: PCR amplification conditions:

1) 42oC for 50 min for reverse transcription 2) 94oC for 3 min

3) 94oC for 30 sec 4) 55oC for 30 sec 5) 72oC for 45 sec Repeat steps 3 to 5 for 35 cycles

6) 72oC for 5 min Store the PCR products at -20 oC.

2. Nested PCR amplification in SupMC plate II with 24 tubes

•••• Open the foil-based self sealing bag labeled “SupMC plate II” (note the plate and the plate user manual card) While keeping the SupMC 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 wells of the plate: PCR reaction buffer II 2.5 µl Taq DNA polymerase (5U/µl) 0.25 µl (provided by user) Molecular grade water to total final volume 21.25 µl

Transfer carefully 1 µl of the primary PCR products with multichannel pipette from each sample and control well in SupMC plate I to the corresponding wells (samples and controls) in the SupMC 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 95oC for 3 min, then

2) 95oC for 30 sec 3) 50oC for 30 sec 4) 72oC for 30 sec Repeat steps 2 to 4 for 25 cycles

5) 72oC for 10 min

Store the PCR products at -20 oC.

C) TCR Clonality assay

1. Make a 4% high resolution (HR) gel Use the high resolution (HR) powder supplied in the kit. If using 4g of the gel, combine with 100 ml 1x TAE buffer in a 500 ml flask. USE CAUTION as the gel will boil vigorously and may bubble out of the flask! Put the gel into the flask, add the TAE, do not swirl, and then heat carefully until the gel is completely melted. Add ethidium bromide (final concentration is 0.5 mg/ml) to the gel mixture and swirl to mix completely. Allow the solution to cool to about 60oC, and then pour into a 12 – 14 cm long gel tray. Add the comb with 25 teeth and allow the gel to solidify at room temperature (at least 20 minutes).

2. Loading the nested PCR products and DNA marker into the gel Add 10 µl of the 10 bp DNA size standard (provided with the kit) to the first well. Combine 10 µl of the nested PCR products from sample (22 Vβ families) and control wells with 1.5 µl of the 6 x loading buffer (provided with the kit) and add to the wells in the gel.

3. Separating DNA fragment by electrophoresis The PCR products are completely separated by electrophoresis in 1 x TAE buffer at 100 V for 1.5 to 2 hours (DNA fragment size of the PCR products of 22 Vβ gene families should be between 100 to 220 bp).

4. Detecting clonality of 22 TCR Vββββ repertoire Clonality of the TCR repertoire can be readily seen under UV light. Usually, there are three types of clonality that can be seen in the gel: Monoclonal – a single clear band Oligoclonal – multiple clear bands Polyclonal – smear with no defined bands

Optional : Screening the CDR3 region

A TA cloning kit (not supplied with the kit) is recommended.

Cut the Vβ family band of interest from the gel and purify the DNA using a gel extraction 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 control

Sample and control cDNA were not amplified

Be sure the Taq polymerase and MMLV reverse transcriptase were added

No signal in the samples but signal appears in positive control

cDNA concentration is too low or no cDNA

Increase cDNA concentration by increasing the starting number of T cells, check cDNA

Signal appears in negative control well

Contamination during PCR preparation

Decontaminate all equipment, including pipettes. Use filtered pipette tips.