Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification

URN to cite this document:

urn:nbn:de:bvb:355-epub-408532 Copy

Durst, Franziska C. ; Grujovic, Ana ; Ganser, Iris ; Hoffmann, Martin ; Ugocsai, Peter ; Klein, Christoph A. ; Czyż, Zbigniew T.

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Date of publication of this fulltext: 31 Oct 2019 08:03

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Details

Item type:	Article
Journal or Publication Title:	PLOS ONE
Publisher:	Public Library of Science
Volume:	14
Number of Issue or Book Chapter:	8
Page Range:	e0216442
Date:	20 August 2019
Institutions:	Medicine > Lehrstuhl für experimentelle Medizin und Therapieverfahren
Projects:	Open Access Publizieren (DFG)
Identification Number:	Value Type 10.1371/journal.pone.0216442 DOI
Dewey Decimal Classification:	600 Technology > 610 Medical sciences Medicine
Status:	Published
Refereed:	Yes, this version has been refereed
Created at the University of Regensburg:	Yes
Item ID:	40853

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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 ...

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 outperforms 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.

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Metadata last modified: 02 Apr 2020 10:46

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