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Related Experiment Videos

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

X Wang1, B D Stollar

  • 1Department of Biochemistry, Tufts University School of Medicine and the Sackler School of Graduate Biomedical Sciences, 136 Harrison Avenue, Boston, MA 02111, USA.

Journal of Immunological Methods
|October 18, 2000
PubMed
Summary

This study details a single-cell reverse transcription polymerase chain reaction (RT-PCR) method for analyzing human immunoglobulin V region gene expression. This technique offers a fast, unbiased approach to studying B cell antibody repertoires.

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Area of Science:

  • Immunology
  • Molecular Biology
  • Genetics

Background:

  • Understanding human immunoglobulin V region usage is crucial for B cell repertoire analysis.
  • Existing methods for V region analysis can introduce bias due to variable mRNA levels.
  • A need exists for a precise and efficient method to study single B cell V region expression.

Purpose of the Study:

  • To describe a protocol for single-cell reverse transcription polymerase chain reaction (RT-PCR) applied to human immunoglobulin V region usage.
  • To provide a method that avoids biases associated with traditional cDNA library construction.
  • To enable detailed analysis of V region gene expression at the single-cell level.

Main Methods:

  • Peripheral blood mononuclear cells (PBMC) are isolated and stained with anti-CD19 antibody.

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  • B cells are sorted by flow cytometry, with single cells deposited into individual tubes.
  • Single-cell cDNA synthesis is followed by nested PCR amplification of antibody variable regions (VH and/or VL).
  • PCR products are sequenced and aligned to germline and Genbank databases.

    • Main Results:

    • The protocol enables direct sequencing and analysis of V region genes from single human B cells.
    • Nested PCR enhances specificity and quantity of V region PCR products.
    • This method provides a fast and convenient way to analyze V region gene expression without library construction bias.

    Conclusions:

    • Single-cell RT-PCR is a powerful tool for unbiased analysis of human immunoglobulin V region usage.
    • The method facilitates the study of B cell antibody repertoires at an unprecedented resolution.
    • Generated PCR products are suitable for cloning into expression vectors for recombinant protein domain production.