Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards.

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Bulk gene expression analysis masks individual cell variability.
• Single-cell analysis is crucial for understanding gene expression noise, gene correlations, and cellular responses.
• Existing single-cell methods like RNA FISH and RNA-Seq have limitations in transcript number, cost, or throughput.

Purpose of the Study:

• To develop and describe a novel procedure for quantifying gene expression in individual mammalian cells.
• To enable the measurement of up to 96 distinct gene transcripts per single cell.
• To provide a cost-effective and high-throughput alternative for single-cell transcriptomic analysis.

Main Methods:

• Single mammalian cells were isolated and sorted using fluorescence-activated cell sorting (FACS) into lysis buffer.
• Messenger RNA (mRNA) was reverse-transcribed and amplified.
• Gene expression was quantified using a microfluidic real-time PCR system capable of performing 96 qPCR assays simultaneously.
• PCR amplicon standards were generated for absolute transcript quantification.

Main Results:

• A procedure for measuring the expression of up to 96 genes in single mammalian cells was successfully established.
• The method allows for the quantification of a higher number of distinct transcripts compared to RNA FISH.
• The described approach offers a lower cost per transcript compared to RNA-Seq.

Conclusions:

• The developed method provides a powerful tool for high-throughput single-cell gene expression analysis.
• This technique facilitates a deeper understanding of cellular heterogeneity and responses.
• It represents a valuable advancement in single-cell transcriptomics, balancing cost and information content.