Human immunoglobulin variable region gene analysis by single cell RT-PCR

• Published in: Journal of immunological methods 2000-10, Vol.244 (1), p.217-225
• Main Authors: Wang, Xiaowei, Stollar, B.David
• Format: Article
• Language: English
• Subjects: Adult; Aged; B cell; B-Lymphocytes - cytology; B-Lymphocytes - immunology; Biological and medical sciences; CD19; cDNA; DNA, Complementary - genetics; Fetal Blood - cytology; Fetal Blood - immunology; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Gene Expression; Gene Rearrangement, B-Lymphocyte - immunology; Genes, Immunoglobulin - genetics; Humans; Immunoglobulin genes; Immunoglobulin Variable Region - blood; Immunoglobulin Variable Region - genetics; Leukocytes, Mononuclear - cytology; Molecular immunology; mRNA; PCR; Repertoire; Reverse Transcriptase Polymerase Chain Reaction; Techniques; V regions
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(00)00260-X

This protocol describes application of single cell reverse transcription polymerase chain reaction (RT-PCR) to the study of human immunoglobulin V region usage. The procedure begins with separation of peripheral blood mononuclear cells (PBMC) from human blood. The PBMC are stained with the B cell selective marker, anti-CD19. Stained B cells are sorted by flow cytometry and deposited, consecutively, one cell into each of an array of tubes. cDNA for one or more antibody variable regions (VH and/or VL) is synthesized with a primer (or primers) complementary to sequence(s) within the constant region (Cμ, Cγ, Cκ and/or Cλ). The cDNA is used as template for PCR amplification with gene or gene family specific primers. A second PCR is then performed with two nested primers to increase both the specificity and quantity of V region PCR products. The purified PCR products are sequenced directly and aligned to V region germline database and the Genbank database. Single cell RT-PCR is a fast and convenient way to analyze V region gene expression. It avoids the bias that may be introduced into V region cDNA library construction by the presence of highly variable levels of mRNA in different cells. The PCR products are obtained in quantities that can be cloned into bacterial expression vectors for production of recombinant V region protein domains.