Global preamplification simplifies targeted mRNA quantification

Kroneis, Thomas; Jonasson, Emma; Andersson, Daniel; Dolatabadi, Soheila; Ståhlberg, Anders

doi:10.1038/srep45219

Cited by 22 publications

(29 citation statements)

References 35 publications

(48 reference statements)

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“…Statistical analysis was performed using Prism version 8.2.1 (GraphPad Software Inc., La Jolla, CA, USA). For library quality assessment, preamplification was performed for a limited number of cycles (24 cycles for single cells and 18 cycles for 128 cells, respectively) using the cDNA quantification protocol, omitting melting curve analysis, as cycling beyond the exponential phase can introduce biases for downstream analyses [25]. As a control, the same samples were analyzed without adding SYBR Green I to the reaction.…”

Section: Total Polyadenylated Rna Analysismentioning

confidence: 99%

Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity

Jonasson

Andersson

Dolatabadi

et al. 2020

Cells

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Single-cell analysis enables detailed molecular characterization of cells in relation to cell type, genotype, cell state, temporal variations, and microenvironment. These studies often include the analysis of individual genes and networks of genes. The total amount of RNA also varies between cells due to important factors, such as cell type, cell size, and cell cycle state. However, there is a lack of simple and sensitive methods to quantify the total amount of RNA, especially mRNA. Here, we developed a method to quantify total mRNA levels in single cells based on global reverse transcription followed by quantitative PCR. Standard curve analyses of diluted RNA and sorted cells showed a wide dynamic range, high reproducibility, and excellent sensitivity. Single-cell analysis of three sarcoma cell lines and human fibroblasts revealed cell type variations, a lognormal distribution of total mRNA levels, and up to an eight-fold difference in total mRNA levels among the cells. The approach can easily be combined with targeted or global gene expression profiling, providing new means to study cell heterogeneity at an individual gene level and at a global level. This method can be used to investigate the biological importance of variations in the total amount of mRNA in healthy as well as pathological conditions.

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Section: Total Polyadenylated Rna Analysismentioning

confidence: 99%

Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity

Jonasson

Andersson

Dolatabadi

et al. 2020

Cells

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“…Each of the available WTA technologies displays unique strengths and weaknesses reflected by differences in detection sensitivity (13, 14). Notably, available WTA strategies do not always provide an overview over the full-length transcripts of a single cell but instead are biased towards specific loci (7, 16) or the ends of the RNA molecules (17–19) limiting downstream analyses to either quantification of selected genes only (targeted analysis) or counting of mRNA molecules, thus limiting the ability to assess transcript diversity or mutational state analysis. The selection of an optimal technique for gene expression analysis in single cells depends on the specific research question, number of samples and genes to be analyzed, as well as the required sensitivity and costs.…”

Section: Introductionmentioning

confidence: 99%

“…In contrast, data analysis for qPCR such as relative and absolute quantification methods (25, 26), are well established, standardized and do not require a high level of expertise (27). Moreover, protocols for preparing qPCR samples are simpler and result in higher sensitivity and reproducibility as compared to NGS-based approaches (7, 27). Importantly, single-cell qPCR workflows exhibit high levels of reliability and wide and dynamic detection ranges, making them exceptionally well-suited for targeted gene expression analyses in singles cells, where sensitivity is essential and the amount of target genes is low (7, 27).…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, protocols for preparing qPCR samples are simpler and result in higher sensitivity and reproducibility as compared to NGS-based approaches (7, 27). Importantly, single-cell qPCR workflows exhibit high levels of reliability and wide and dynamic detection ranges, making them exceptionally well-suited for targeted gene expression analyses in singles cells, where sensitivity is essential and the amount of target genes is low (7, 27). Therefore, the present study aimed to develop a single-cell qPCR assay to quantify gene expression changes in single cells, specifically in patient-derived DCCs.…”

Section: Introductionmentioning

confidence: 99%

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

Durst

Grujovic

Ganser

et al. 2019

Preprint

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2Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer 3 cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we 4 developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell 5 cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) 6 protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-7 amplification of WTA products, quantification of amplified cDNA yields and final qPCR 8 quantification, to identify the most reliable and accurate workflow for quantitation of gene 9 expression of the ERBB2 gene in DCCs. We found that absolute quantification outperforms 10 relative quantification. We then validated the performance of our method on single cells of 11 established breast cancer cell lines displaying distinct levels of HER2 protein. The different 12 protein levels were faithfully reflected by transcript expression across the tested cell lines 13 thereby proving the accuracy of our approach. Finally, we applied our method on patient-derived 14 breast cancer DCCs. Here, we were able to measure ERBB2 expression levels in all HER2-15 positive DCCs. In addition, we could detect ERBB2 transcript expression even in HER2-negative 16 DCCs, suggesting post-transcriptional mechanisms of HER2 loss in anti-HER2-treated DCCs. In 17 summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of 18 ERBB2 in DCCs. 19 66(27). Moreover, protocols for preparing qPCR samples are simpler and result in higher 67 sensitivity and reproducibility as compared to NGS-based approaches (7, 27). Importantly, 68 single-cell qPCR workflows exhibit high levels of reliability and wide and dynamic detection 69 ranges, making them exceptionally well-suited for targeted gene expression analyses in singles 70 cells, where sensitivity is essential and the amount of target genes is low (7, 27). Therefore, the 71 present study aimed to develop a single-cell qPCR assay to quantify gene expression changes in 72 single cells, specifically in patient-derived DCCs. We established a workflow comprised of 73 single-cell WTA, re-amplification of single-cell cDNA, post-WTA normalization of cDNA 74 quantities and qPCR-based data analysis. The new assay provides means for measuring 75 expression levels of individual pre-selected genes in WTA products generated from single cells 76 in an accurate and reliable fashion.77 6 78 Materials and methods 79 Cell lines 80 BT-474 (ACC 64) and MCF-7 (ACC 115) breast cancer cell lines were obtained from German 81 Collection of Microorganisms and Cell Cultures (DSMZ). MCF-10A (CRL-10317), a non-82 tumorigenic mammary epithelial cell line was obtained from American Type Culture Collection 83 (ATCC). ZR-75-1 (CRL1500, ATCC) and MDA-MB-453 (ACC 65, DSMZ) cells were 84 purchased from DSMZ. The identity of all cell lines was confirmed by DNA finger printing 85 analysis utilizing the GenePrint® 10 System (Promega). B...

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“…An effective method for increasing detection sensitivity is the addition of a preamplification step before gene expression analysis through qPCR (4). Many studies confirm the accuracy of the preamplification step, which is applied on cDNA template synthesized by reverse transcription (5)(6)(7)(8). Furthermore, the preamplification procedure is a modified version of conventional PCR.…”

Section: Introductionmentioning

confidence: 99%

Increasing detection sensitivity of low copy number transcripts through preamplification of cDNA molecules

Dimaras

Tasinov

Ivanova

et al. 2017

SSM

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With the advances in the field of molecular biology and its applications in the clinical practice during the recent years, it has become crucial to perform molecular analyses on limited amount of tissue obtained through biopsies. Additionally, tissue is fixed in formalin and further embedded in paraffin (FFPE), a procedure which causes extensive degradation of nucleic acids, mainly RNAs. Furthermore, when studying gene expression profiles of a set of genes, which present physiologically low expression, the number of transcripts is low and cannot be detected. In this study we focus on amplifying effectively cDNA molecules synthesized from small amounts of initial RNA before analyzing the expression levels through quantitative polymerase chain reaction (qPCR). By introducing the preamplification step in the procedure, we achieved highly efficacious detection and quantification of expression levels of low-expression genes.

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Global preamplification simplifies targeted mRNA quantification

Cited by 22 publications

References 35 publications

Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity

Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity

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

Increasing detection sensitivity of low copy number transcripts through preamplification of cDNA molecules

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