Bead-based RNA multiplex panels for biomarker detection in oncology samples

Scerri, Jeanesse; Baldacchino, Shawn; Saliba, Christian; Scerri, Christian; Grech, Godfrey

doi:10.1016/j.ymeth.2018.10.008

Cited by 4 publications

(4 citation statements)

References 34 publications

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“…The QuantiGene TM 2.0 Plex Assay platform (ThermoFisher Scientific, Waltham, MA, USA) allows for the effective multiplexing of up to 80 targets with an ELISA-like workflow in a 96-well format, which reduces workload and costs. A crude lysate can be used as a sample, which obviates the isolation steps performed, for example, in PCR methods and thus makes the method less error and contamination prone [ 38 ]. The robust and low-complexity workflow together with the durable and relatively cheap reader device also allows for its use in places with limited resources.…”

Section: Discussionmentioning

confidence: 99%

Human Papillomavirus E7 and p16INK4a mRNA Multiplexed Quantification by a QuantiGeneTM Proof-of-Concept Assay Sensitively Detects Infection and Cervical Dysplasia Severity

et al. 2023

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Background: Persistent infection with human papillomavirus (HPV) can lead to cervical cancer (CxCa). During the progression to CxCa, the expression of HPV oncogenes E6 and E7 is upregulated. In turn, cellular proteins such as p16INK4a are also modulated. The combined detection of HPV oncogenes and cellular biomarkers indicative for dysplasia could be informative and convey better specificity than the current HPV tests that cannot discriminate transient infection from dysplastic changes. Methods: The QuantiGeneTM 2.0 Plex Assay platform was chosen for the effective multiplexing and quantitative detection of seven HPV-E7 mRNA targets (HPV6, 16, 18, 31, 45, 59, and 68) and the cellular mRNA of p16INK4a as a biomarker for HPV-induced transformation. Actin-beta (ACTB) and hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) were included as reference markers. Sequences for the specific capture and detector probes were customized and developed by ThermoFisher and formulated as a QuantiGene proof-of-concept (QG-POC) plex-set. The crude lysates of the HPV-positive cervical cancer cell lines CaSki (HPV16), HeLa (HPV18), MRHI-215 (HPV45), Erin59 (HPV59), ME180 (HPV68), and the HPV-negative cell line C33A, as well as liquid-based cytology smear samples (n = 441) were analyzed. The study was a proof-of-concept evaluating the feasibility of the platform. Logistic regression and receiver operating characteristic (ROC) analyses were performed to test for the sensitivity and specificity of HPV detection and dysplastic stage discrimination. Results: A QG-POC assay specifically and sensitively detects the HPV-E7 mRNA of seven different genotypes with an assay linearity between 20 and 13,000 cells. Cellular mRNA was detected from the crude lysates of cell lines and of cellular material from clinical liquid-based cytology smear samples. By combining HPV-E7 and p16INK4a expression normalized to ACTB, high-grade dysplasia (HCIN) and invasive cervical cancer (CxCa) were detectable, discriminable, and correlated to the biomarker expression strength. The ROC analysis from the multivariate logistic regression model including HPV-E7 and p16 INK4a resulted in an AUC of 0.74, at the optimal cut-off (sensitivity: 70.4%; specificity: 66.0%) for HCIN detection. CxCa was detected with an AUC of 0.77 (sensitivity: 81.8%, specificity: 77.4%). Conclusions: The QG-POC assay is sufficiently sensitive to detect and quantify HPV-E7 and cellular mRNA species. Multiplexing allows the specific detection of at least 10 analytes in a single reaction. Determining the abundance of E7 and p16INK4a transcripts when normalized to ACTB is informative about the presence of cervical dysplasia and potentially discriminates between low-grade and high-grade dysplasia and invasive cervical cancer. Further studies including more HPV genotypes and biomarkers are warranted.

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Section: Discussionmentioning

confidence: 99%

Human Papillomavirus E7 and p16INK4a mRNA Multiplexed Quantification by a QuantiGeneTM Proof-of-Concept Assay Sensitively Detects Infection and Cervical Dysplasia Severity

et al. 2023

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“…For Her2-positive breast cancer, however, there is no FDA-approved gene panel to date for the clinical prediction of response to trastuzumab-containing treatment regimes. The use of bead-based, multiplex RNA ( 33 ) and protein ( 34 ) assays has shown effectiveness in medium- to high-throughput cancer biomarker discovery and detection.…”

Section: Introductionmentioning

confidence: 99%

PKC-mediated phosphorylation and activation of the MEK/ERK pathway as a mechanism of acquired trastuzumab resistance in HER2-positive breast cancer

Scerri

Schäfer-Ruoff

et al. 2022

Front. Endocrinol.

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Protein expression, activation and stability are regulated through inter-connected signal transduction pathways resulting in specific cellular states. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins (phospho-protein profile) in key signal transduction pathways, in breast cancer cellular models of trastuzumab resistance. To this effect, we utilized a multiplex, bead-based protein assay, DigiWest®, to measure around 100 proteins and protein modifications using specific antibodies. The main advantage of this methodology is the quantification of multiple analytes in one sample, utilising input volumes of a normal western blot. The intrinsically trastuzumab-resistant cell line JIMT-1 showed the largest number of concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, β catenin stabilization by inhibitory phosphorylation of GSK3β, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance cellular models. The overexpression of upstream RAS/RAF, however, was confined to JIMT 1; meanwhile, in a cellular model of acquired trastuzumab resistance generated in this study (T15), entry into the ERK pathway seemed to be mostly mediated by PKCα activation. This is a novel observation and merits further investigation that can lead to new therapeutic combinations in HER2-positive breast cancer with acquired therapeutic resistance.

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“…Fluorescent bead–based analysis using branched DNA (bDNA) technology is an emerging technique for gene expression profiling (Collins et al, 1997 ; Scerri et al, 2019 ; Tsongalis, 2006 ). In contrast to the aforementioned gene expression platforms, the bDNA technique uses signal amplification rather than target amplification to measure messenger RNAs (mRNAs); and it does not rely on RNA extraction, cDNA synthesis, and PCR amplification.…”

Section: Introductionmentioning

confidence: 99%

“…In contrast to the aforementioned gene expression platforms, the bDNA technique uses signal amplification rather than target amplification to measure messenger RNAs (mRNAs); and it does not rely on RNA extraction, cDNA synthesis, and PCR amplification. The Invitrogen QuantiGene Plex Assay (QGP; Thermo Fisher Scientific) is one such assay, incorporating bDNA technology for the direct measurement of RNA transcripts (Scerri et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool

Karengera

Bao

Bovee

et al. 2022

Enviro Toxic and Chemistry

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Gene expression profiling in Caenorhabditis elegans has been demonstrated to be a potential bioanalytical tool to detect the toxic potency of environmental contaminants. The RNA transcripts of genes responding to toxic exposure can be used as biomarkers for detecting these toxins. For routine application in environmental quality monitoring, an easy-to-use multiplex assay is required to reliably quantify expression levels of these biomarkers. In the present study, a bead-based assay was developed to fingerprint gene expression in C. elegans by quantitating messenger RNAs (mRNAs) of multiple target genes directly from crude nematode lysates, circumventing RNA extraction and purification steps. The assay uses signal amplification rather than target amplification for direct measurement of toxin-induced RNA transcripts. Using a 50-gene panel, the expression changes of four candidate reference genes and 46 target mRNAs for various contaminants and wastewaters were successfully measured, and the expression profiles indicated the type of toxin present. Moreover, the multiplex assay response was in line with previous results obtained with more time-consuming reverse-transcription quantitative polymerase chain reaction and microarray analyses. In addition, the transcriptomic profiles of nematodes exposed to wastewater samples and extracts prepared from tissues of swimming crabs were evaluated. The profiles indicated the presence of organic pollutants. The present study illustrates the successful development of a multiplex fluorescent bead-based approach using nematode C. elegans crude lysates for gene expression profiling of target RNAs. This method can be used to routinely fingerprint the presence of toxic contaminants in environmental samples and to identify the most biologically active fraction of the contaminant mixture in a toxicity identification and evaluation approach. Environ Toxicol Chem 2023;42:130-142.

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Bead-based RNA multiplex panels for biomarker detection in oncology samples

Cited by 4 publications

References 34 publications

Human Papillomavirus E7 and p16INK4a mRNA Multiplexed Quantification by a QuantiGeneTM Proof-of-Concept Assay Sensitively Detects Infection and Cervical Dysplasia Severity

Human Papillomavirus E7 and p16INK4a mRNA Multiplexed Quantification by a QuantiGeneTM Proof-of-Concept Assay Sensitively Detects Infection and Cervical Dysplasia Severity

PKC-mediated phosphorylation and activation of the MEK/ERK pathway as a mechanism of acquired trastuzumab resistance in HER2-positive breast cancer

A Multiplex Gene Expression Assay for Direct Measurement of RNA Transcripts in Crude Lysates of the Nematode Caenorhabditis elegans Used as a Bioanalytical Tool

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