High throughput analysis of TCR-beta rearrangement and gene expression in single T cells.
Analysis of T-cell receptor beta chain (TCR-beta) rearrangement is essential to investigate T-cell responses in human autoimmune diseases, infection and cancer. Since the TCR-beta locus contains 55 variable (V) region gene segments, multiple assays have been necessary to determine TCR-beta rearrangements of individual T cells. We established a seminested rtPCR method for single T-cell analysis with two sets of degenerate primers covering 76 and 24% of the TCR-Vbeta genes, respectively. The specificity of the approach was validated by screening cDNAs obtained from T-cell clones (TCC) with defined TCR-beta rearrangement. We applied the method successfully to profile TCR-beta rearrangement of single T cells sorted from body fluids or dissected tissue. Concomitant analysis of other gene transcripts allowed determining phenotype and function of TCR-beta-defined single T cells. Our fast, cost-efficient and high throughput approach will facilitate studies on T-cell responses in human diseases.
Zhou
Dun
D
Neuroimmunology Group, Department of Neurology, Heinrich Heine-University, Duesseldorf, Germany.
Srivastava
Rajneesh
R
Grummel
Verena
V
Cepok
Sabine
S
Hartung
Hans-Peter
HP
Hemmer
Bernhard
B
D001932:Brain Neoplasms / Q000737:chemistry / Q000235:genetics; D002469:Cell Separation; D017931:DNA Primers / Q000737:chemistry; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D042282:Microdissection; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000737:chemistry*
D017931:DNA Primers; D012333:RNA, Messenger