Tutorial: Guidelines for Single-Cell RT-qPCR

Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.

Automated summary

Reverse transcription quantitative PCR (RT-qPCR) is a valuable tool for understanding gene expression, particularly in single-cell analysis. This article discusses the limitations of single-cell collection methods, emphasizes the importance of reverse transcription, provides recommendations for preamplification and primer design, and summarizes essential data processing steps. The detailed protocol in the appendix allows any researcher to conduct high-standard scRT-qPCR measurements.