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.

• Keywords
• RT-qPCR, gene expression, preamplification, quantitative PCR, reverse transcription, sample collection, single cell,
• MeSH
• Single-Cell Analysis methods   standards   MeSH
• Real-Time Polymerase Chain Reaction methods   standards   MeSH
• Humans MeSH
• Reverse Transcription genetics   MeSH
• Sensitivity and Specificity MeSH
• Gene Expression Profiling methods   standards   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

In this study, we investigated the influence of metformin (MF) on proliferation and viability of adipose-derived stromal cells isolated from horses (EqASCs). We determined the effect of metformin on cell metabolism in terms of mitochondrial metabolism and oxidative status. Our purpose was to evaluate the metformin effect on cells derived from healthy horses (EqASCHE) and individuals affected by equine metabolic syndrome (EqASCEMS). The cells were treated with 0.5 μM MF for 72 h. The proliferative activity was evaluated based on the measurement of BrdU incorporation during DNA synthesis, as well as population doubling time rate (PDT) and distribution of EqASCs in the cell cycle. The influence of metformin on EqASC viability was determined in relation to apoptosis profile, mitochondrial membrane potential, oxidative stress markers and BAX/BCL-2 mRNA ratio. Further, we were interested in possibility of metformin affecting the Wnt3a signalling pathway and, thus, we determined mRNA and protein level of WNT3A and β-catenin. Finally, using a two-tailed RT-qPCR method, we investigated the expression of miR-16-5p, miR-21-5p, miR-29a-3p, miR-140-3p and miR-145-5p. Obtained results indicate pro-proliferative and anti-apoptotic effects of metformin on EqASCs. In this study, MF significantly improved proliferation of EqASCs, which manifested in increased synthesis of DNA and lowered PDT value. Additionally, metformin improved metabolism and viability of cells, which correlated with higher mitochondrial membrane potential, reduced apoptosis and increased WNT3A/β-catenin expression. Metformin modulates the miRNA expression differently in EqASCHE and EqASCEMS. Metformin may be used as a preconditioning agent which stimulates proliferative activity and viability of EqASCs.

• Keywords
• adipose-derived stromal cells, equine metabolic syndrome, metformin,
• MeSH
• Apoptosis drug effects   MeSH
• beta Catenin metabolism   MeSH
• Cell Cycle drug effects   MeSH
• Horses MeSH
• Cells, Cultured MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Metabolic Syndrome drug therapy   pathology   veterinary   MeSH
• Metformin pharmacology   therapeutic use   MeSH
• MicroRNAs genetics   metabolism   MeSH
• Mitochondria drug effects   metabolism   MeSH
• Multipotent Stem Cells cytology   MeSH
• Oxidation-Reduction MeSH
• Cell Proliferation drug effects   MeSH
• Wnt3A Protein metabolism   MeSH
• Cell Separation * MeSH
• Adipose Tissue cytology   MeSH
• Cell Survival drug effects   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta Catenin MeSH
• Metformin MeSH
• MicroRNAs MeSH
• Wnt3A Protein MeSH