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 repr...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-09-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/10/10/2607 |
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| author | Daniel Zucha Mikael Kubista Lukas Valihrach |
| author_facet | Daniel Zucha Mikael Kubista Lukas Valihrach |
| author_sort | Daniel Zucha |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T06:39:16Z |
| format | Article |
| id | doaj.art-2db13fca13654d6195b462610e2d7546 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T06:39:16Z |
| publishDate | 2021-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-2db13fca13654d6195b462610e2d75462023-11-22T17:46:19ZengMDPI AGCells2073-44092021-09-011010260710.3390/cells10102607Tutorial: Guidelines for Single-Cell RT-qPCRDaniel Zucha0Mikael Kubista1Lukas Valihrach2Laboratory of Gene Expression, Institute of Biotechnology CAS, 252 50 Vestec, Czech RepublicLaboratory of Gene Expression, Institute of Biotechnology CAS, 252 50 Vestec, Czech RepublicLaboratory of Gene Expression, Institute of Biotechnology CAS, 252 50 Vestec, Czech RepublicReverse 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.https://www.mdpi.com/2073-4409/10/10/2607single cellsample collectionreverse transcriptionpreamplificationquantitative PCRgene expression |
| spellingShingle | Daniel Zucha Mikael Kubista Lukas Valihrach Tutorial: Guidelines for Single-Cell RT-qPCR Cells single cell sample collection reverse transcription preamplification quantitative PCR gene expression |
| title | Tutorial: Guidelines for Single-Cell RT-qPCR |
| title_full | Tutorial: Guidelines for Single-Cell RT-qPCR |
| title_fullStr | Tutorial: Guidelines for Single-Cell RT-qPCR |
| title_full_unstemmed | Tutorial: Guidelines for Single-Cell RT-qPCR |
| title_short | Tutorial: Guidelines for Single-Cell RT-qPCR |
| title_sort | tutorial guidelines for single cell rt qpcr |
| topic | single cell sample collection reverse transcription preamplification quantitative PCR gene expression |
| url | https://www.mdpi.com/2073-4409/10/10/2607 |
| work_keys_str_mv | AT danielzucha tutorialguidelinesforsinglecellrtqpcr AT mikaelkubista tutorialguidelinesforsinglecellrtqpcr AT lukasvalihrach tutorialguidelinesforsinglecellrtqpcr |