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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.<\/jats:p>","DOI":"10.3390\/cells10102607","type":"journal-article","created":{"date-parts":[[2021,10,1]],"date-time":"2021-10-01T10:55:40Z","timestamp":1633085740000},"page":"2607","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":39,"title":["Tutorial: Guidelines for Single-Cell RT-qPCR"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2469-1315","authenticated-orcid":false,"given":"Daniel","family":"Zucha","sequence":"first","affiliation":[{"name":"Laboratory of Gene Expression, Institute of Biotechnology CAS, 252 50 Vestec, Czech Republic"},{"name":"Department of Informatics and Chemistry, Faculty of Chemical Technology, University of Chemistry and Technology, 166 28 Prague, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mikael","family":"Kubista","sequence":"additional","affiliation":[{"name":"Laboratory of Gene Expression, Institute of Biotechnology CAS, 252 50 Vestec, Czech Republic"},{"name":"TATAA Biocenter AB, 411 03 Gothenburg, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6704-4337","authenticated-orcid":false,"given":"Lukas","family":"Valihrach","sequence":"additional","affiliation":[{"name":"Laboratory of Gene Expression, Institute of Biotechnology CAS, 252 50 Vestec, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,9,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1038\/s41591-018-0223-3","article-title":"Identification of evolutionarily conserved gene networks mediating neurodegenerative dementia","volume":"25","author":"Swarup","year":"2019","journal-title":"Nat. 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