Open AccessA simple way to increase recovery of the expressed VH and VL genes in single-sorted human B cells
Author(s) -
Sergey V. Guselnikov,
Tatya. Belovezhets,
Sergey V. Kulemzin,
Andrey A. Gorchakov,
Alexander V. Taranin
Publication year - 2019
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/btn-2019-0079
Subject(s) - gene , cloning (programming) , biology , microbiology and biotechnology , monoclonal antibody , genetics , antigen , polymerase chain reaction , antibody , computational biology , computer science , programming language
Cloning VH and VL 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 VH and VL genes in 90–100% of single sorted human B cells.