Development of TaqMan-based real-time PCR based on ψ gene for quantitative detection of CAR-T cells.
Anal Biochem
; 694: 115626, 2024 Nov.
Article
em En
| MEDLINE
| ID: mdl-39032527
ABSTRACT
Chimeric-antigen-receptor-T (CAR-T) have heralded a paradigm shift in the landscape of cancer immunotherapy. Retrovirus-mediated gene transfer serves to deliver the specific CAR expressing cassette into T cells across a spectrum of basic research and clinical contests in cancer therapy. However, it is necessary to devise a precise and validated quantitative methodology tailored to the diverse CAR constructs. In the investigation, a TaqMan real-time qPCR method was developed, utilizing primers targeting ψ gene sequence. This method offers a swift, sensitive, reproducible, and accurate tool for evaluating retroviral copy numbers at the integrated DNA level. Importantly, the established qPCR exhibits no cross-reactivity with non-transduced T cells or tissues. The regression equation characterizing TaqMan real-time PCR dynamics is y = -3.3841x + 41.402 (R2 = 0.999), showing an amplification efficiency of 97.47 %. Notably, the established qPCR method achieves a minimum detection of 43.1 copies/µL. Furthermore, both intra- and inter-group discrepancies remain below 4 %, underscoring the good repeatability of the established method. Our in vitro and in vivo results also support its sensitivity, specificity, and stability. Consequently, this method offers researchers with a cost-effective tool to quantify CAR copies both in vitro and in vivo.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Linfócitos T
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Reação em Cadeia da Polimerase em Tempo Real
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Receptores de Antígenos Quiméricos
Limite:
Animals
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Humans
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2024
Tipo de documento:
Article