Quantitative PCR Analysis of DNA, RNAs, and Proteins in the Same Single Cell
Open accessAbstract
BACKGROUND: The single cell represents the basic unit of all organisms. Most investigations have been performed on large cell populations, but understanding cell dynamics and heterogeneity requires single-cell analysis. Current methods for single-cell analysis generally can detect only one class of analytes. METHODS: Reverse transcription and the proximity ligation assay were coupled with quantitative PCR and used to quantify any combination of DNA, mRNAs, microRNAs (miRNAs), noncoding RNAs (ncRNAs), and proteins from the same single cell. The method was used on transiently transfected human cells to determine the intracellular concentrations of plasmids, their transcribed mRNAs, translated proteins, and downstream RNA targets. RESULTS: We developed a whole-cell lysis buffer to release unfractionated DNA, RNA, and proteins that would not degrade any detectable analyte or inhibit the assay. The dynamic range, analytical sensitivity, and specificity for quantifying DNA, mRNAs, miRNAs, ncRNAs, and proteins were shown to be accurate down to the single-cell level. Correlation studies revealed that the intracellular concentrations of plasmids and their transcribed mRNAs were correlated only moderately with translated protein concentrations (Spearman correlation coefficient, 0.37 and 0.31, respectively; P < 0.01). In addition, an ectopically expressed gene affected the correlations between analytes and this gene, which is related to gene regulation. CONCLUSIONS: This method is compatible with most cell-sampling approaches, and generates output for the same parameter for all measured analytes, a feature facilitating comparative data analysis. This approach should open up new avenues in molecular diagnostics for detailed correlation studies of multiple and different classes of analytes at the single-cell level.
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Many multi-omics studies co-profiling protein and mRNA rely on predetermined curated antibody panels, losing novel and unexpected proteomic information.
“Many multi-omics studies, in which protein and mRNA are co-profiled, rely on the use of predetermined, curated antibody panels to confer single-cell phenotypes, thereby losing novel and unexpected proteomic information63–66.”
From The technological landscape and applications of single-cell multi-omics (2023) · cites this paper as [66]
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- Authors
- Ståhlberg, A., Thomsen, C., Ruff, D., & Åman, P.
- Affiliations
- Tataa Biocenter (Sweden) (SE)
- University of Gothenburg (SE)
- Applied Biosystems, Part of Life Technologies, Foster City, CA
- Corresponding author
- Ståhlberg, Anders
- Venue
- Clinical Chemistry
- Publisher
- Oxford University Press (OUP)
- Published
- 2012-12-01
- Volume
- 58
- Issue
- 12
- Pages
- 1682-1691
- ISSN
- 0009-9147 (ISSN-L)
- 1530-8561
- OpenAlex
- W2151946432
- Cited by
- 78
- References
- 41
- Citations by year
- 2026: 3 · 2025: 1 · 2024: 3 · 2023: 6 · 2022: 3 · 2021: 6 · 2020: 3 · 2019: 6 · 2018: 5 · 2017: 10 · 2016: 8 · 2015: 10 · 2014: 6 · 2013: 7 · 2012: 1
- FWCI
- 3.08
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model (vor)
- Locations
- Clinical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Swedish Research Council (2367)
- Swedish Society for Medical Research
- Johan Jansson Foundation for Cancer Research
- Assar Gabrielssons Research Foundation
- Wilhelm and Martina Lundgren Foundation for Scientific Research
- Swedish Cancer Society
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Keywords
- single-cell analysis, proximity ligation assay, real-time PCR, reverse transcription, mRNA quantification, microRNA quantification, protein quantification, plasmid transfection, gene expression correlation, cellular heterogeneity, molecular diagnosis
- MeSH terms
- Buffers, DNA, Humans, Proteins, Recombinant Fusion Proteins, RNA, RNA, Messenger, Polymerase Chain Reaction, RNA, Untranslated, RNA-Binding Protein FUS, MicroRNAs, Cell Line, Tumor, Green Fluorescent Proteins, Single-Cell Analysis
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