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A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

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Evaluation of single-cell sorting accuracy using antibody-derived tag-based qPCR.

Xiaoshan Shi¹, Woodrow E Lomas², Aaron Middlebrook²

¹Applied Research & Technology, Medical & Scientific Affairs, BD Biosciences, Milpitas, California, USA.

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|August 12, 2024

Summary

This study introduces a new assay to confirm single-cell sorting accuracy using antibody-derived tags (ADTs) and qPCR. The method also enables proteomic profiling of individual cells and cell lysates.

Keywords:

TaqMan™ qPCR antibody‐derived tags (ADTs)single‐cell sorting (index sorting)

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Published on: August 22, 2019

Area of Science:

Biotechnology
Proteomics
Cell Biology

Background:

Single-cell sorting via fluorescence-activated cell sorting (FACS) is crucial for applications like single-cell sequencing.
Limited assays exist to verify the proteomic identity and sorting accuracy of individual cells post-isolation.

Purpose of the Study:

To develop a novel assay for confirming protein expression in sorted single cells.
To establish a method for proteomic profiling at the single-cell level.
To validate the accuracy of single-cell sorting.

Main Methods:

Co-staining cells with antibody-derived tags (ADTs) and fluorescent antibodies.
Amplifying ADT oligo sequences from sorted single cells using multiplex TaqMan™ quantitative polymerase chain reaction (qPCR).
Applying the assay to characterize protein expression in whole cell lysates.

Main Results:

The developed assay successfully confirmed protein expression in sorted single cells.
The assay demonstrated efficiency in profiling proteomic features at the single-cell level.
The high sensitivity of TaqMan™ qPCR allowed detection of protein expression from small cell numbers.

Conclusions:

The ADT-based qPCR assay is effective for confirming single-cell sorting accuracy.
This method provides a sensitive approach for proteomic characterization of both single cells and cell lysates.
The assay enhances the reliability of single-cell isolation techniques.