对EBV 和CMV特异性人CD8+T淋巴细胞的TCRβ克隆型分析

Nening M. Nanlohy; Dan Koning; Esther D. Quakkelaar; Debbie van Baarle

doi:10.21769/BioProtoc.1606

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Peer-reviewed

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

NN Nening M. Nanlohy

DK Dan Koning

EQ Esther D. Quakkelaar

DB Debbie van Baarle email

发布: 2015年10月05日第5卷第19期 DOI: 10.21769/BioProtoc.1606 浏览次数: 8020

评审: Kathrin SutterMarielle CavroisAnonymous reviewer(s)

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Cover of Journal of Virology, featuring study using the protocol.

Jan 2015

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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

Nuclease-free 1.5 ml tubes (Sarstedt, catalog number: 72692005 )
RNAlater (Life Technologies, catalog number: AM7020 )
Note: Currently, it is “Thermo Fisher Scientific, Ambion^TM, catalog number: AM7020”.
FACS-sorted CD8⁺ T cells in RNAlater (Koning et al., 2013)
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^®)
LIVE/DEAD Viability dye (Thermo Fisher Scientific, Invitrogen^TM)
µMACS mRNA isolation kit (Miltenyi Biotec, catalog number: 130-075-201 )
SMARTer Pico PCR cDNA synthesis kit (Takara Bio Company, Clontech, catalog number: 634928 )
dNTPs 25 mM each (Life Technologies, catalog number: R1121 )
Recombinant RNasin Ribonuclease Inhibitor 20 U/μl (Promega Corporation, catalog number: N2511 )
Superscript II RNase H^- Reverse transcriptase 200 U/μl (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 18064014 )
Note: This kit also contains dithiothreitol (DTT, 100 mM).
Nucleospin Extract II, gel and PCR clean up (MACHEREY-NAGEL GmbH & Co, catalog number: MN740609.250 )
Molecular grade H₂O (Sigma-Aldrich, catalog number: W4502-1L )
Advantage 2 Polymerase Mix (Takara Bio Company, Clontech, catalog number: 639201 )
Molecular grade agarose (Thermo Fisher Scientific, Fisher BioReagents^®, catalog number: 10688973 )
SYBR^® Gold Nucleic Acid Gel Stain (Life Technologies, catalog number: S-11494 )
Note: Currently, it is “Thermo Fisher Scientific, Invitrogen^TM, catalog number: S-11494”.
1 kb Plus DNA ladder (Life Technologies, catalog number: 10787-018 )
Note: Currently, it is “Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10787-018 ”.
pGEM-T Easy Vector System kit I (Promega Corporation, catalog number: A1360 )
Chemically competent E. coli DH5α (Home-made)
AmpliTaq DNA Polymerase with buffer II (Life Technologies, catalog number: N8080153 )
Note: Currently, it is “Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: N8080153”.
Big Dye Terminator v3.1 cycle kit (Life Technologies, catalog number: 4337455 )
Note: Currently, it is “Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4337455”.
RNase-inactivator (RNase Away) (MP Biomedicals)

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).

10x Universal Primer mix (see Recipes)
50 mM DTT (see Recipes)

Equipment

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

Procedure

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如何引用

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.