Durst, Franziska C. and Grujovic, Ana and Ganser, Iris and Hoffmann, Martin and Ugocsai, Peter and Klein, Christoph A. and Czyz, Zbigniew T. (2019) Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification. PLOS ONE, 14 (8): e0216442. ISSN 1932-6203,
Full text not available from this repository. (Request a copy)Abstract
Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-amplification of WTA products, quantification of amplified cDNA yields and final qPCR quantification, to identify the most reliable and accurate workflow for quantitation of gene expression of the ERBB2 gene in DCCs. We found that absolute quantification out-performs relative quantification. We then validated the performance of our method on single cells of established breast cancer cell lines displaying distinct levels of HER2 protein. The different protein levels were faithfully reflected by transcript expression across the tested cell lines thereby proving the accuracy of our approach. Finally, we applied our method to breast cancer DCCs of a patient undergoing anti-HER2-directed therapy. Here, we were able to measure ERBB2 expression levels in all HER2-protein-positive DCCs. In summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of ERBB2 in DCCs.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | REAL-TIME PCR; STOCHASTIC GENE-EXPRESSION; RNA-SEQ; MOLECULES; FREQUENCY; TOOL; |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für experimentelle Medizin und Therapieverfahren |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 31 Mar 2020 13:29 |
| Last Modified: | 31 Mar 2020 13:29 |
| URI: | https://pred.uni-regensburg.de/id/eprint/26419 |
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