Institute of Molecular & Cellular Biology SB RAS, Novosibirsk, 630090, Russia.
Novosibirsk State University, Novosibirsk, 630090, Russia.
Biotechniques. 2019 Oct;67(4):184-187. doi: 10.2144/btn-2019-0079. Epub 2019 Aug 14.
Cloning and genes from individual antigen-specific B cells is an attractive approach for producing monoclonal antibodies of the desired specificity. Current RT-PCR protocols, however, result in the successful identification of VH and VL gene pairs in about half of the sorted cells. Here, we demonstrate that single-cell RT-PCR is likely affected by stochastic factors, and that running PCRs in triplicate results in successful amplification of the expressed and genes in 90-100% of single sorted human B cells.
从单个抗原特异性 B 细胞中克隆和基因是生产所需特异性单克隆抗体的一种有吸引力的方法。然而，目前的 RT-PCR 方案只能成功鉴定出约一半已分选细胞中的 VH 和 VL 基因对。在这里，我们证明单细胞 RT-PCR 可能受到随机因素的影响，并且三管 PCR 运行可使 90-100%的单个分选人 B 细胞中表达的 和 基因成功扩增。
