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

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.