Tutorial: Guidelines for Single-Cell RT-qPCR
Abstract
:1. Introduction
2. Sample collection
2.1. Preparation of Single-Cell Suspension
2.2. Single-Cell Collection
2.3. RNA Extraction and DNase Treatment
2.4. Direct Lysis
2.5. Storage Conditions
2.6. Small Bulk Analysis
3. Reverse Transcription
3.1. RT-Associated Variables
3.2. RTase Selection
3.3. Priming Strategy
3.4. RT Additives
3.5. Temperature Profile
3.6. Setting RT Reaction
3.7. Quality Control
4. Preamplification
4.1. Targeted vs. Global Preamplification
4.2. Reaction Parameters
4.3. Validation
4.4. Setting preAMP Reaction
5. Quantitative PCR
5.1. Assay Design
5.2. Assay Validation
5.3. Limit of Detection and Limit of Quantification
5.4. Setting the qPCR Reaction
5.5. Quality Control
6. Data Analysis
6.1. Single-Cell Data Specifications
6.2. Data Pre-Processing
6.3. Descriptive Analysis and Data Visualization
7. Conclusions
Supplementary Materials
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Zucha, D.; Kubista, M.; Valihrach, L. Tutorial: Guidelines for Single-Cell RT-qPCR. Cells 2021, 10, 2607. https://doi.org/10.3390/cells10102607
Zucha D, Kubista M, Valihrach L. Tutorial: Guidelines for Single-Cell RT-qPCR. Cells. 2021; 10(10):2607. https://doi.org/10.3390/cells10102607
Chicago/Turabian StyleZucha, Daniel, Mikael Kubista, and Lukas Valihrach. 2021. "Tutorial: Guidelines for Single-Cell RT-qPCR" Cells 10, no. 10: 2607. https://doi.org/10.3390/cells10102607
APA StyleZucha, D., Kubista, M., & Valihrach, L. (2021). Tutorial: Guidelines for Single-Cell RT-qPCR. Cells, 10(10), 2607. https://doi.org/10.3390/cells10102607