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TCRβ Clonotype Analysis of EBV and CMV-specific Human CD8+ T Cells
对EBV 和CMV特异性人CD8+T淋巴细胞的TCRβ克隆型分析   

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Abstract

This protocol describes the quantification of all expressed T-cell antigen receptor (TCR) gene products within sorted (by flow cytometry) EBV and CMV-specific memory CD8+ T-cell populations using a template-switch anchored reverse transcription polymerase chain reaction (RT-PCR).

Materials and Reagents

  1. Nuclease-free 1.5 ml tubes (Sarstedt, catalog number: 72692005 )
  2. RNAlater (Life Technologies, catalog number: AM7020 )
    Note: Currently, it is “Thermo Fisher Scientific, AmbionTM, catalog number: AM7020”.
  3. FACS-sorted CD8+ T cells in RNAlater (Koning et al., 2013)
  4. T-cell markers CD3 (PerCP) (BD), CD8 (AmCyan/V500) (BD) and pMHC-tetramer (APC/PE) (home-made), and the dump markers CD14 and CD19 (both BioLegend, Pacific Blue®)
  5. LIVE/DEAD Viability dye (Thermo Fisher Scientific, InvitrogenTM)
  6. µMACS mRNA isolation kit (Miltenyi Biotec, catalog number: 130-075-201 )
  7. SMARTer Pico PCR cDNA synthesis kit (Takara Bio Company, Clontech, catalog number: 634928 )
  8. dNTPs 25 mM each (Life Technologies, catalog number: R1121 )
  9. Recombinant RNasin Ribonuclease Inhibitor 20 U/μl (Promega Corporation, catalog number: N2511 )
  10. Superscript II RNase H- Reverse transcriptase 200 U/μl (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18064014 )
    Note: This kit also contains dithiothreitol (DTT, 100 mM).
  11. Nucleospin Extract II, gel and PCR clean up (MACHEREY-NAGEL GmbH & Co, catalog number: MN740609.250 )
  12. Molecular grade H2O (Sigma-Aldrich, catalog number: W4502-1L )
  13. Advantage 2 Polymerase Mix (Takara Bio Company, Clontech, catalog number: 639201 )
  14. Molecular grade agarose (Thermo Fisher Scientific, Fisher BioReagents®, catalog number: 10688973 )
  15. SYBR® Gold Nucleic Acid Gel Stain (Life Technologies, catalog number: S-11494 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: S-11494”.
  16. 1 kb Plus DNA ladder (Life Technologies, catalog number: 10787-018 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 10787-018 ”.
  17. pGEM-T Easy Vector System kit I (Promega Corporation, catalog number: A1360 )
  18. Chemically competent E. coli DH5α (Home-made)
  19. AmpliTaq DNA Polymerase with buffer II (Life Technologies, catalog number: N8080153 )
    Note: Currently, it is “Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: N8080153”.
  20. Big Dye Terminator v3.1 cycle kit (Life Technologies, catalog number: 4337455 )
    Note: Currently, it is “Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4337455”.
  21. RNase-inactivator (RNase Away) (MP Biomedicals)
  22. Primers (can be ordered from any company that provides custom made DNA oligo’s):
    SMART II oligo
    5’-AAGCAGTGGTATCAACGCAGAGTACGCGGG*-3’
    Universal
    Primer Long

    5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’
    Universal
    Primer Short

    5’-CTAATACGACTCACTATAGGGC-3’
    MBC2
    5’-TGCTTCTGATGGCTCAAACACAGCGACCT-3’
    M13 Forward
    5’-TTTTCCCAGTCACGAC-3’
    M13 Reverse
    5’-CAGGAAACAGCTATGAC-3’
    Oligo d(T)25
    5’-d(T)25VN**-3’
    *The final GGG bases in this oligo are RNA bases
    **V = A, C, or G. N = A, C, G, or T
    Note: Preferably, order primers with high purity (HPLC purified).
  23. 10x Universal Primer mix (see Recipes)
  24. 50 mM DTT (see Recipes)

Equipment

  1. FACSAria III flow cytometer (BD)
  2. Heating blocks (42 °C and 70 °C) (VWR International)
  3. T-M-AR, DNA/RNA UV-cleaner box (Laminar flow hood/ UVC) (Biosan, catalog number: BS-040104-A06 )
  4. Standard table top Eppendorf centrifuge (for use at room temperature and 4 °C)
  5. PCR thermo cycler

Procedure

  1. FACS sort of antigen-specific T cells
    1. In the studies conducting this protocol, EBV and CMV-specific memory CD8+ T-cell populations were sorted using pMHC class I tetramers on a FACSAria III flow cytometer. A high fidelity staining strategy was used, comprising the specific T-cell markers CD3, CD8 and pMHC-tetramer, and the dump markers CD14 and CD19 to exclude any contamination during the sort from monocytes and B cells, respectively. In addition, a LIVE/DEAD Viability dye was included in the cell staining. For details on the specific staining, see Koning et al. (2013). Tetramer-positive populations usually comprised 0.1-1.0% of CD8+ T cells and in general 500-5,000 antigen-specific cells were isolated.
      Note: Although the specific fluorochromes noted here were used in Koning et al. (2013), we have performed a similar staining strategy using different fluorochromes in other studies. Similarly, although this protocol specifies the analysis of EBV and CMV-specific T cells, there are no restrictions for the analysis of T cells directed against other species/antigens.
      To obtain a complete and accurate view of the TCR composition of an antigen-specific T-cell population, make sure to sort a sufficient number of cells (at least 500-1,000 cells, ideally 5,000 cells or even more) at high purity (by including dump markers and a viability dye, as described above). Sort cells directly in RNAlater and store at -80 °C.
  2. mRNA isolation
    1. Isolate mRNA using the µMACS mRNA Isolation kit, according to the manufacturer’s instructions. Elute with 30 µl preheated elution buffer.
    2. Store mRNA at -80 °C or proceed with cDNA synthesis.
  3. cDNA generation
    1. Incubate 5.5 µl mRNA and 0.5 µl Oligo d(T)25 (25 mM) at 2 min at 70 °C followed by 2 min at 42 °C.
      Note: Proceed as fast as possible after this step. Time can be won by preparing the mastermix below during the initial incubation. Ideally, preparation of the mastermix is finished at the same time as the incubation.
    2. Add:
      2.2 µl
      5x First Strand buffer
      from SMARTer cDNA synthesis kit
      0.4 µl
      DTT (50 mM)

      0.4 µl
      dNTPs (25 mM)

      1 µl
      SMART II Oligo (12 µM)
      from SMARTer cDNA synthesis kit or custom made*
      0.5 µl
      RNasin (20 U/µl)

      1 µl
      SuperScript II Reverse Transcriptase (200 U/µl)

    3. Incubate 2 h at 42 °C.
      Note: *Whilst a custom made SMART II oligo can be cost-reducing, it may perform less than the oligo that is included in the SMARTer cDNA synthesis kit.
  4. Purification of cDNA
    Purify the cDNA using the Nucleospin Extract II Gel and PCR Clean Up kit. Follow the manufacturer’s protocol, with the exceptions described below.
    Add to the cDNA:
    77 µl H2O
    13 µl buffer NTI
    (Total volume: 100 µl)
    A single wash step with 650 µl buffer NT3 suffices. Elute in 25 µl buffer NE.
    Preferably, continue directly to the RACE (rapid amplification of cDNA ends) TCRβ PCR or store the purified cDNA at -80 °C.
  5. RACE TCRβ-PCR
    RACE PCR is performed using TCR-specific and anchor-complementary primers.
    Prepare a PCR mastermix of all reagents except for the template. Prepare enough for a duplicate of each sample and one negative control (water instead of template). Small, artificial variations in clonotype composition can be caused by the PCR. Therefore, when a very accurate examination of the T-cell repertoire is desired (for example, in a longitudinal analysis), it is advised to perform a duplicate PCR that can be pooled after agarose gel electrophoresis (step 5).
    1. Per sample

      5 µl
      10x Advantage 2 SA PCR buffer
      10 µl
      10x Universal Primer mix (forward primers)
      1 µl
      MBC2 (25 µM, reverse primer)
      1 µl
      dNTPs (10 mM)
      1 µl
      50x Advantage 2 Polymerase mix
      12 µl
      Purified cDNA
      20 µl
      H2O
      50 µl
      Total volume
    2. Run reactions in a thermocycler under the following conditions:
      1 cycle
      95 °C
      30 sec
      5 cycles:
      95 °C
      15 sec

      72 °C
      2 min
      5 cycles:
      95 °C
      15 sec

      70 °C
      30 sec

      72 °C
      2 min
      30 cycles:
      95 °C
      15 sec

      68 °C
      30 sec

      72 °C
      2 min

      4 °C

      Store the TCRβ products at -20 °C.
      Note: Do not store at 4 °C, as the A overhang of the PCR products will degrade over time.
  6. Isolation of Vβ products
    1. Run an electrophoresis on a 1% agarose gel, using a 1 kb DNA ladder to determine the size of the fragments. Leave an empty well between each sample to minimize the chance of cross-contamination. A band at 500-600 bp should be visible. Carefully excise the band of interest and transfer the gel to a clean 1.5 ml microcentrifuge tube.
    2. Extract the amplified TCR products from the gel using the NucleoSpin Extract II Gel and PCR Clean Up kit, according to the manufacturer’s instructions. Store samples at -20 °C.
      Note: Perform the extra wash steps described in the protocol and also use preheated (37 °C) wash buffer NT3. Elute in 20 µl buffer NE.
  7. Ligation of Vβ product into plasmid and sequencing
    Amplified TCR transcripts are ligated into the pGEM-T Easy vector. Prepare 15 µl ligation reactions, using 5 µl of sample, 7.5 µl Rapid ligation buffer, 1 µl pGEM-T Easy Vector, and 1.5 µl T4 DNA ligase. Incubate overnight (16-24 h) at 4 °C, then transform into chemically competent E. coli DH5α bacteria using 7.5 µl of the ligation mixture. Pick 96 single transformed bacterial clones to amplify using M13 primers. Use the following PCR mix per sample (=per bacterial colony):
    2.5 µl
    AmpliTaq PCR Buffer II
    0.2 µl
    M13 Forward primer (25 µM)
    0.2 µl
    M13 Reverse primer (25 µM)
    0.5 µl
    dNTPs (10 mM)
    0.125 µl
    AmpliTaq DNA polymerase
    3.0 µl
    MgCl2 (25 mM)
    18.475 µl
    H2O
    25 µl
    Total volume
    Use the following cycling conditions:
    1 cycle
    95 °C
    30 sec
    40 cycles:
    95 °C
    15 sec

    57 °C
    30 sec

    72 °C
    90 sec
    Hold:
    4 °C

    Assess the efficiency of amplification on a 1% agarose gel. Sanger sequencing is performed using the Big Dye Terminator v3.1 Cycle kit and samples are sent to a high throughput facility to be sequenced by capillary electrophoresis.

Representative data

Example of a TCRβ-PCR:


Figure 1. A duplicate TCRβ-PCR was performed on 3 different donors. A 1 kb Plus DNA ladder was used. The correct PCR product is ±550 bp long. Note the presence of PCR products other than the desired one, which is a common side effect of SMART cDNA synthesis.

Notes

  1. The success of this protocol depends on high quality mRNA and a “clean” working environment (DNase/RNase-free and the presence of PCR-grade disposables) throughout the procedure. Contamination must be minimized by cleaning with 10% bleach solution and/or an RNase-inactivator.
  2. Aside from poor mRNA quality/quantity, many factors can cause the PCR to fail. One of the easier factors to exclude as a cause is the reagents you work with. Make sure all stocks are clean and prepared correctly, using molecular grade water to prepare dilutions. Also, do not expose reagents to room temperature for longer than is necessary.
  3. Furthermore, use fresh reagents. Especially SuperScript II Reverse Transcriptase is sensitive for losing its activity over time. Ideally, all reagents are less than one year old. While a sample with high input RNA (for example, 1.0 x 106 cells from a T-cell clone) may not suffer from suboptimal reagents, samples with few cells (500-5,000) are very sensitive to the solutions used.

Recipes

  1. 10x Universal Primer mix
    Prepare a mix containing 0.4 μM Universal Primer Long and 2.0 μM Universal primer Short
  2. 50 mM DTT
    Dilute the 100 mM DTT stock (from the SuperScript II Reverse Transcriptase kit) 1:1 with molecular grade H2O

Acknowledgments

This protocol was adapted from the previously published studies (Douek et al., 2002; Quigley et al., 2011).

References

  1. Douek, D. C., Betts, M. R., Brenchley, J. M., Hill, B. J., Ambrozak, D. R., Ngai, K. L., Karandikar, N. J., Casazza, J. P. and Koup, R. A. (2002). A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape. J Immunol 168(6): 3099-3104.
  2. Koning, D., Costa, A. I., Hoof, I., Miles, J. J., Nanlohy, N. M., Ladell, K., Matthews, K. K., Venturi, V., Schellens, I. M., Borghans, J. A., Kesmir, C., Price, D. A. and van Baarle, D. (2013). CD8+ TCR repertoire formation is guided primarily by the peptide component of the antigenic complex. J Immunol 190(3): 931-939.
  3. Quigley, M. F., Almeida, J. R., Price, D. A. and Douek, D. C. (2011). Unbiased molecular analysis of T cell receptor expression using template-switch anchored RT-PCR. Curr Protoc Immunol Chapter 10: Unit10 33.

简介

该方案描述了使用模板转换锚定的(通过流式细胞术)EBV和CMV特异性记忆CD8 + T细胞群体中所有表达的T细胞抗原受体(TCR)基因产物的定量 逆转录聚合酶链反应(RT-PCR)。

材料和试剂

  1. 无核酸酶1.5ml管(Sarstedt,目录号:72692005)
  2. RNAlater(Life Technologies,目录号:AM7020)
    注意:目前,"Thermo Fisher Scientific,Ambion TM ,目录号:AM7020" />
  3. RNAlater中的FACS分选的CD8 + sup/+ T细胞(Koning等人,2013)
  4. T细胞标记物CD3(PerCP)(BD),CD8(AmCyan/V500)(BD)和pMHC-四聚体(APC/PE)(自制),以及倾倒标记物CD14和CD19(BioLegend, sup>?)
  5. LIVE/DEAD活力染料(Thermo Fisher Scientific,Invitrogen TM
  6. μMACSmRNA分离试剂盒(Miltenyi Biotec,目录号:130-075-201)
  7. SMARTer Pico PCR cDNA合成试剂盒(Takara Bio Company,Clontech,目录号:634928)
  8. dNTP各25mM(Life Technologies,目录号:R1121)
  9. 重组RNasin核糖核酸酶抑制剂20U /μl(Promega Corporation,目录号:N2511)
  10. 上标II RNA酶H 逆转录酶200U /μl(Thermo Fisher Scientific,Invitrogen TM,目录号:18064014)
    注意:此试剂盒还含有二硫苏糖醇(DTT,100 mM)。
  11. Nucleospin Extract II,凝胶和PCR清洗(MACHEREY-NAGEL GmbH& Co,目录号:MN740609.250)
  12. 分子级H 2 O(Sigma-Aldrich,目录号:W4502-1L)
  13. Advantage 2聚合酶混合物(Takara Bio Company,Clontech,目录号:639201)
  14. 分子级琼脂糖(Thermo Fisher Scientific,Fisher BioReagents ,目录号:10688973)
  15. SYBR Gold Nucleic Acid Gel Stain(Life Technologies,目录号:S-11494)
    注意:目前,"赛默飞世尔科技有限公司
  16. 1kb Plus DNA ladder(Life Technologies,目录号:10787-018)
    注意:目前,"Thermo Fisher Scientific,Invitrogen TM ,目录号:10787-01 > 8"。
  17. pGEM-T Easy Vector System试剂盒I(Promega Corporation,目录号:A1360)
  18. 具有化学能力的大肠杆菌DH5α(自制)
  19. AmpliTaq DNA聚合酶与缓冲液II(Life Technologies,目录号:N8080153)
    注意:目前,"Thermo Fisher Scientific,Applied Biosystems TM N8080153"。
  20. Big Dye Terminator v3.1循环试剂盒(Life Technologies,目录号:4337455)
    注意:目前,"Thermo Fisher Scientific,Applied Biosystems TM ,目录号:4337455" br />
  21. 核糖核酸酶灭活剂(RNase Away)(MP Biomedicals)
  22. 引物(可从提供定制DNA寡核苷酸的任何公司订购):
    SMART II寡聚物
    5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG * -3'
    通用底漆长
    5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'
    通用底漆短
    5'-CTAATACGACTCACTATAGGGC-3'
    MBC2
    5'-TGCTTCTGATGGCTCAAACACAGCGACCT-3'
    M13转发
    5'-TTTTCCCAGTCACGAC-3'
    M13反向
    5'-CAGGAAACAGCTATGAC-3'
    Oligo d(T) 25
    5'-d(T) 25 VN ** - 3'
    *该寡核苷酸中的最终GGG碱基是RNA碱基
    ** V = A,C或G. N = A,C,G或T
    注意:最好订购高纯度的引物(HPLC纯化)。
  23. 10x通用底漆混合物(参见配方)
  24. 50 mM DTT(参见配方)

设备

  1. FACSAria III流式细胞仪(BD)
  2. 加热块(42℃和70℃)(VWR International)
  3. T-M-AR,DNA/RNA UV清洁盒(Laminair流动罩/UVC)(Biosan,目录号:BS-040104-A06)
  4. 标准台式Eppendorf离心机(在室温和4°C下使用)
  5. PCR热循环仪

程序

  1. FACS种类的抗原特异性T细胞
    1. 在进行该方案的研究中,EBV和CMV特异性记忆 使用pMHC I类四聚体在a上分离CD8 + T细胞群体 FACSAria III流式细胞仪。使用高保真染色策略, ?包括特异性T细胞标记物CD3,CD8和pMHC-四聚体,和 转储标记CD14和CD19,以排除任何污染 分别从单核细胞和B细胞分选。此外,LIVE/DEAD 活细胞染色被包括在细胞染色中。有关的详细信息 特异性染色,参见Koning等人(2013)。四聚体阳性 群体通常包含0.1-1.0%的CD8 + T细胞和一般 分离500-5,000个抗原特异性细胞。
      注意:虽然 特异性荧光染料用于Koning等人(2013),我们 已经使用不同的荧光染料进行了sim染色策略 ?在其他研究。同样,虽然这个协议指定了 分析EBV和CMV特异性T细胞,没有限制 分析针对其他物种/抗原的T细胞。
      至 获得完整和准确的TCR组成的视图 抗原特异性T细胞群体,确保分选足够 细胞数(至少500-1,000个细胞,理想地5,000个细胞或甚至) 更多)高纯度(通过包括倾倒标记和活力染料, 如上所述)。单元格直接在RNAlater和存储在-80°C。
  2. mRNA分离
    1. 使用μMACSmRNA分离试剂盒分离mRNA,根据 制造商的说明。用30μl预热的洗脱缓冲液洗脱
    2. 将mRNA储存于-80℃或进行cDNA合成。
  3. cDNA生成
    1. 在70℃2分钟孵育5.5μlmRNA和0.5μlOligo d(T)25分(25mM),然后在42℃孵育2分钟。
      注意:此步骤后尽可能快。时间可以赢 在初始孵育期间制备下面的mastermix。理想情况下, 准备混音的同时完成 孵化。
    2. 添加:
      2.2微升
      5x第一串缓冲区
      来自SMARTer cDNA合成试剂盒
      0.4μl
      DTT(50mM)

      0.4μl
      dNTPs(25mM)
      1微升
      SMART II Oligo(12μM)
      从SMARTer cDNA合成试剂盒或定制*
      0.5μl
      RNasin(20U /μl)

      1微升
      SuperScript II Reverse Transcriptase(200 U /μl)

    3. 在42°C孵育2小时。
      注意:*虽然定制的SMART II寡核苷酸可以降低成本,但它可以 ?表现小于包含在SMARTer cDNA中的寡核苷酸 合成试剂盒。
  4. cDNA纯化
    使用Nucleospin Extract II凝胶和PCR清理工具包纯化cDNA。遵循制造商的协议,除了下面描述的。
    添加到cDNA:
    77μlH 2 O * 13μl缓冲液NTI
    (总体积:100μl)
    用650μl缓冲液NT3进行单次洗涤步骤就足够了。在25μl缓冲液NE中洗脱 优选地,继续直接进行RACE(cDNA末端的快速扩增)TCRβPCR或将纯化的cDNA储存在-80℃。
  5. RACETCRβ-PCR
    使用TCR特异性和锚 - 互补引物进行RACE PCR。
    准备所有试剂除模板外的PCR主混合物。准备足够的副本的每个样品和一个阴性对照(水代替模板)。克隆型组成的小的,人为的变化可以由PCR引起。因此,当需要非常准确地检测T细胞库(例如,在纵向分析中)时,建议进行在琼脂糖凝胶电泳(步骤5)后可以合并的重复PCR。
    1. 每个样本

      5微升
      10x Advantage 2 SA PCR缓冲液
      10微升
      10x通用引物混合物(正向引物)
      1微升
      MBC2(25μM,反向引物)
      1微升
      dNTPs(10mM)
      1微升
      50x Advantage 2聚合酶混合物
      12微升
      纯化的cDNA
      20微升
      H sub 2 O
      50微升
      总量
    2. 在热循环仪中在以下条件下进行反应:
      1个周期
      95°C
      30秒
      5个周期:
      95°C
      15秒

      72℃
      2分钟
      5个周期:
      95°C
      15秒

      70℃
      30秒

      72℃
      2分钟
      30个循环:
      95°C
      15秒

      68°C
      30秒

      72℃
      2分钟

      4°C

      将TCRβ产品储存在-20°C 注意:不要在4℃保存,因为PCR产物的A突出端会随时间降解。

  6. Vβ产物的分离
    1. 在1%琼脂糖凝胶上电泳,使用1 kb DNA梯度 确定片段的大小。在每个之间留下一个空的井 样品以最小化交叉污染的机会。一个乐队在500-600 bp应该可见。仔细切除感兴趣的带和转移 ?将凝胶置于干净的1.5ml微量离心管中
    2. 提取 使用NucleoSpin Extract II凝胶从凝胶中扩增TCR产物 和PCR Clean Up试剂盒,根据制造商的说明。 将样品储存在-20°C。
      注意:执行额外的 sh步骤 描述在协议和也使用预热(37℃)洗涤缓冲液 NT3。在20μl缓冲液NE中洗脱。
  7. 将Vβ产物连接到质粒和测序中
    将扩增的TCR转录物连接到pGEM-T Easy载体中。使用5μl样品,7.5μl快速连接缓冲液,1μlpGEM-T Easy Vector和1.5μlT4 DNA连接酶制备15μl连接反应物。在4℃孵育过夜(16-24小时),然后使用7.5μl连接混合物转化到化学感受态大肠杆菌DH5α细菌中。挑选96个单个转化的细菌克隆,使用M13引物扩增。每个样品使用以下PCR混合物(=每个细菌菌落):
    2.5μl
    AmpliTaq PCR Buffer II
    0.2μl
    M13正向引物(25μM)
    0.2μl
    M13反向引物(25μM)
    0.5μl
    dNTPs(10mM)
    0.125μl
    AmpliTaq DNA聚合酶
    3.0μl
    MgCl 2(25mM)
    18.475微升
    H sub 2 O
    25μl
    总量
    使用以下骑车条件:
    1个周期
    95°C
    30秒
    40个周期:
    95°C
    15秒

    57℃
    30秒

    72℃
    90秒
    保持:
    4°C

    评估在1%琼脂糖凝胶上的扩增效率。 Sanger测序使用Big Dye Terminator v3.1循环试剂盒进行,样品送至高通量设备,通过毛细管电泳进行测序。

代表数据

TCRβ-PCR的实施例:


图1.对3个不同供体进行重复TCRβ-PCR。使用1kb Plus DNA梯。正确的PCR产物长度为±550bp。注意除了所需的PCR产物的存在,这是SMART cDNA合成的常见副作用。

笔记


  1. 这个协议的成功取决于高质量的mRNA和"干净" 工作环境(DNase/RNase-free和PCR级的存在 一次性用品)。污染必须最小化 通过用10%漂白溶液和/或RNA酶灭活剂清洁。
  2. 除了差的mRNA质量/数量,许多因素可以导致PCR 失败。作为原因排除的更容易的因素之一是试剂 ?你使用。确保所有的库存清洁,准备正确, 使用分子级水制备稀释液。此外,不要暴露 试剂到室温的时间长于必要。
  3. 此外,使用新鲜的试剂。特别是SuperScript II Reverse 转录酶对于失去其随时间的活性敏感。理想情况下, 所有试剂均小于一岁。而具有高输入的样本 RNA(例如,来自T细胞克隆的1.0×10 6个细胞) 不遭受次优试剂,具有少量细胞的样品(500-5,000) 对所使用的解决方案非常敏感。


食谱

  1. 10x通用引物混合物
    准备含有0.4μM通用引物长和2.0μM通用引物短的混合物
  2. 50 mM DTT
    用分子级H 2 O 1稀释100mM DTT储液(来自SuperScript II Reverse Transcriptase试剂盒)1:1

致谢

该方案改编自以前公开的研究(Douek等人,2002; Quigley等人,2011)。

参考文献

  1. Douek,D.C.,Betts,M.R.,Brenchley,J.M.,Hill,B.J.,Ambrozak,D.R.,Ngai,K.L.,Karandikar,N.J.,Casazza,J.P.and Koup,R.A。(2002)。 一种分析HIV特异性T细胞反应的特异性,克隆性和频率的新方法揭示一种用于控制病毒逃逸的潜在机制。 J Immunol 168(6):3099-3104。
  2. Koning,D.,Costa,AI,Hoof,I.,Miles,JJ,Nanlohy,NM,Ladell,K.,Matthews,KK,Venturi,V.,Schellens,IM,Borghans,JA,Kesmir, ,DA和van Baarle,D。(2013)。 CD8 + TCR汇辑谱形成主要由抗原复合物。 J Immunol 190(3):931-939。
  3. Quigley,M.F.,Almeida,J.R.,Price,D.A。和Douek,D.C。(2011)。 使用模板转换锚定的RT-PCR对T细胞受体表达的无偏分子分析。 rr Protoc Immunol
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引用:Nanlohy, N. M., Koning, D., Quakkelaar, E. D. and Baarle, D. v. (2015). TCRβ Clonotype Analysis of EBV and CMV-specific Human CD8+ T Cells. Bio-protocol 5(19): e1606. DOI: 10.21769/BioProtoc.1606.
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