29 results found
Qureshi M, Kaluarachchi M, Vorkas P, et al., 2018, Metabolic Profiling of High-Risk Carotid Atherosclerosis, Vascular Annual Meeting of the Society-for-Vascular-Surgery (SVS), Publisher: MOSBY-ELSEVIER, Pages: E236-E236, ISSN: 0741-5214
Vorkas PA, 2018, Expanding lipidome coverage using MS/MS-aided untargeted data-independent RP-UPLC-TOF-MSE acquisition, Bioanalysis, Vol: 10, Pages: 307-319, ISSN: 1757-6180
Lipid function and importance in disease are being rediscovered due to modern advancements in chemicalanalysis. RP–UPLC–TOF–MSE is now the lipidomics tool of choice and can provide the demanded specificityfor detecting the great diversity of the lipidome. It can offer simplicity, rapidity, robustness and highthroughputness, without the need for further optimization in current sample preparation protocols. Thismethod can cover the major lipid categories with the ability to detect several corresponding subclasses.It can deliver adequate information for deciphering fatty chain length, unsaturation and regioisomerism.It has enabled the detection of a vast number of lipids, of which more than 250 are reported here. Theselipids were detected from applications in a variety of biological matrices and species.
Vorkas PA, Abellona U MR, Li JV, 2018, Tissue Multiplatform-Based Metabolomics/Metabonomics for Enhanced Metabolome Coverage., Pages: 239-260
The use of tissue as a matrix to elucidate disease pathology or explore intervention comes with several advantages. It allows investigation of the target alteration directly at the focal location and facilitates the detection of molecules that could become elusive after secretion into biofluids. However, tissue metabolomics/metabonomics comes with challenges not encountered in biofluid analyses. Furthermore, tissue heterogeneity does not allow for tissue aliquoting. Here we describe a multiplatform, multi-method workflow which enables metabolic profiling analysis of tissue samples, while it can deliver enhanced metabolome coverage. After applying a dual consecutive extraction (organic followed by aqueous), tissue extracts are analyzed by reversed-phase (RP-) and hydrophilic interaction liquid chromatography (HILIC-) ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy. This pipeline incorporates the required quality control features, enhances versatility, allows provisional aliquoting of tissue extracts for future guided analyses, expands the range of metabolites robustly detected, and supports data integration. It has been successfully employed for the analysis of a wide range of tissue types.
Rahman S, Vorkas P, Morrison D, et al., 2017, ACUTE DIETARY SATURATED FAT INTAKE CAN SUPPRESS THE INFLAMMATORY RESPONSE IN HUMAN CIRCULATING FOAMY MONOCYTES, 85th Congress of the European-Atherosclerosis-Society (EAS), Publisher: Elsevier, Pages: E116-E116, ISSN: 0021-9150
Anwar MA, Adesina-Georgiadis KN, Spagou K, et al., 2017, A comprehensive characterisation of the metabolic profile of varicose veins; implications in elaborating plausible cellular pathways for disease pathogenesis, Scientific Reports, Vol: 7, ISSN: 2045-2322
Metabolic phenotypes reflect both the genetic and environmental factors which contribute to the development of varicose veins (VV). This study utilises analytical techniques to provide a comprehensive metabolic picture of VV disease, with the aim of identifying putative cellular pathways of disease pathogenesis. VV (n = 80) and non-VV (n = 35) aqueous and lipid metabolite extracts were analysed using 600 MHz 1H Nuclear Magnetic Resonance spectroscopy and Ultra-Performance Liquid Chromatography Mass Spectrometry. A subset of tissue samples (8 subjects and 8 controls) were analysed for microRNA expression and the data analysed with mirBase (www.mirbase.org). Using Multivariate statistical analysis, Ingenuity pathway analysis software, DIANALAB database and published literature, the association of significant metabolites with relevant cellular pathways were understood. Higher concentrations of glutamate, taurine, myo-inositol, creatine and inosine were present in aqueous extracts and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in lipid extracts in the VV group compared with non-VV group. Out of 7 differentially expressed miRNAs, spearman correlation testing highlighted correlation of hsa-miR-642a-3p, hsa-miR-4459 and hsa-miR-135a-3p expression with inosine in the vein tissue, while miR-216a-5p, conversely, was correlated with phosphatidylcholine and phosphatidylethanolamine. Pathway analysis revealed an association of phosphatidylcholine and sphingomyelin with inflammation and myo-inositol with cellular proliferation.
Qureshi MI, Vorkas P, Kaluarachchi M, et al., 2017, Biomarker research in thromboembolic stroke, Annual Meeting of the Society-of-Academic-and-Research-Surgery (SARS), Publisher: WILEY, Pages: 12-13, ISSN: 0007-1323
Qureshi MI, Greco M, Vorkas PA, et al., 2017, Application of metabolic profiling to abdominal aortic aneurysm research, Journal of Proteome Research, Vol: 16, Pages: 2325-2332, ISSN: 1535-3893
Abdominal aortic aneurysm (AAA) is a complex disease posing diagnostic and therapeutic challenges. Metabonomics may aid in the diagnosis of AAA, determination of individualized risk, discovery of therapeutic targets, and improve understanding of pathogenesis. A systematic review of the diversity and outcomes of existing AAA metabonomic research has been performed. Original research studies applying metabonomics to human aneurysmal disease are included. Seven relevant articles were identified: four studies were based on plasma/serum metabolite profiling, and three studies examined aneurysmal tissue. Aminomalonic acid, guanidinosuccinic acid, and glycerol emerge as potential plasma biomarkers of large aneurysm. Lipid profiling improves predictive models of aneurysm presence. Patterns of metabolite variation associated with AAA relate to carbohydrate and lipid metabolism. Perioperative perturbations in metabolites suggest differential systemic inflammatory responses to surgery, generating hypotheses for adjunctive perioperative therapy. Significant limitations include small study sizes, lack of correction for multiple testing false discovery rates, and single time-point sampling. Metabolic profiling carries the potential to identify biomarkers of AAA and elucidate pathways underlying aneurysmal disease. Statistically and methodologically robust studies are required for validation, addressing the hiatus in understanding mechanisms of aneurysm growth and developing effective treatment strategies.
Qureshi M, Vorkas P, Kaluarachchi M, et al., 2017, Biomarker research in thromboembolic stroke (BRUITS), Publisher: KARGER, ISSN: 1015-9770
Qureshi MI, Vorkas PA, Coupland AP, et al., 2016, Lessons from metabonomics on the neurobiology of stroke, Neuroscientist, Vol: 23, Pages: 374-382, ISSN: 1073-8584
The application of metabonomic science to interrogate stroke permits the study of metabolite entities, small enough to cross the blood-brain barrier, that provide insight into neuronal dysfunction, and may serve as reservoirs of biomarker discovery. This systematic review examines the applicability of metabolic profiling in ischemic stroke research. Six human studies utilizing metabolic profiling to analyze biofluids from ischemic stroke patients have been included, employing 1H-NMR and/or mass spectrometry to analyze plasma, serum, and/or urine in a targeted or untargeted fashion. Three are diagnostic studies, and one investigates prognostic biomarkers of stroke recurrence following transient ischemic attack. Two studies focus on metabolic distinguishers of depression or cognitive impairment following stroke. Identified biomarkers from blood and urine predominantly relate to homocysteine and folate, branched chain amino acid, and lipid metabolism. Statistical models are well fitted and reproducible, with excellent validation outcomes, demonstrating the feasibility of metabolic profiling to study a complex disorder with multicausal pathology, such as stroke.
Anwar MA, Vorkas PA, Li J, et al., 2016, Prolonged Mechanical Circumferential Stretch Induces Metabolic Changes in Rat Inferior Vena Cava, EUROPEAN JOURNAL OF VASCULAR AND ENDOVASCULAR SURGERY, Vol: 52, Pages: 544-552, ISSN: 1078-5884
Djekic D, Pinto R, Vorkas PA, et al., 2016, Replication of LC-MS untargeted lipidomics results in patients with calcific coronary disease: an interlaboratory reproducibility study, International Journal of Cardiology, Vol: 222, Pages: 1042-1048, ISSN: 1874-1754
BackgroundRecently a lipidomics approach was able to identify perturbed fatty acyl chain (FAC) and sphingolipid moieties that could stratify patients according to the severity of coronary calcification, a form of subclinical atherosclerosis. Nevertheless, these findings have not yet been reproduced before generalising their application. The aim of this study was to evaluate the reproducibility of lipidomics approaches by replicating previous lipidomic findings in groups of patients with calcific coronary artery disease (CCAD).MethodsPatients were separated into the following groups based on their calcium score (CS); no calcification (CS: 0; n = 26), mild calcification (CS: 1–250; n = 27) and severe calcification (CS: > 250; n = 17). Two serum samples were collected from each patient and used for comparative analyses by 2 different laboratories, in different countries and time points using liquid chromatography coupled to mass spectrometry untargeted lipidomics methods.ResultsSix identical metabolites differentiated patients with severe coronary artery calcification from those with no calcification were found by both laboratories independently. Additionally, relative intensities from the two analyses demonstrated high correlation coefficients. Phosphatidylcholine moieties with 18-carbon FAC were identified in lower intensities and 20:4 FAC in higher intensities in the serum of diseased group. Moreover, 3 common sphingomyelins were detected.ConclusionThis is the first interlaboratory reproducibility study utilising lipidomics applications in general and specifically in patients with CCAD. Lipid profiling applications in patients with CCAD are very reproducible in highly specialised and experienced laboratories and could be applied in clinical practice in order to spare patients diagnostic radiation.
Vorkas PA, Shalhoub J, Lewis MR, et al., 2016, Metabolic Phenotypes of Carotid Atherosclerotic Plaques Relate to Stroke Risk – An Exploratory Study, European Journal of Vascular and Endovascular Surgery, Vol: 52, Pages: 5-10, ISSN: 1532-2165
Objectives: Stroke is a major cause of death and disability. The fact that three-quarters of stroke patients will never have previously manifested cerebrovascular symptoms demonstrates the unmet clinical need for new biomarkers able to stratify patient risk and elucidation of the biological dysregulations. In this study, we assess the utility of comprehensive metabolic phenotyping to provide candidate biomarkers that relate to stroke risk in stenosing carotid plaque tissue samples.Design: Carotid plaque tissue samples were obtained from patients with cerebrovascular symptoms of carotid origin (n=5), and asymptomatic patients (n=5). Two adjacent biological replicates were obtained from each tissue.Materials and Methods: Organic and aqueous metabolite extracts were separately obtained and analysed using two ultra performance liquid chromatography coupled to mass spectrometry metabolic profiling methods. Multivariate and univariate tools were utilised for statistical analysis.Results: The two studied groups demonstrated distinct plaque phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the two groups with a strong statistical significance (p=10-4-10-5). Specifically, metabolites related to the eicosanoid pathway (arachidonic acid and arachidonic acid precursors), and three acylcarnitine species (butyrylcarnitine, hexanoylcarnitine and palmitoylcarnitine), intermediates of the β-oxidation, were detected in higher intensities in symptomatic patients. However, metabolites implicated in the process of cell death, a process known to be upregulated in the formation of the vulnerable plaque, were unaffected.Conclusions: Discrimination between symptomatic and asymptomatic carotid plaque tissue is demonstrated for the first time using metabolic profiling technologies. Two biological pathways (eicosanoid and β-oxidation) were implicated and will be further investigated. These results indicate that metabolic
Lamour SD, Gomez-Romero M, Vorkas PA, et al., 2015, Discovery of Infection Associated Metabolic Markers in Human African Trypanosomiasis., PLOS Neglected Tropical Diseases, Vol: 9, ISSN: 1935-2735
Human African trypanosomiasis (HAT) remains a major neglected tropical disease in Sub-Saharan Africa. As clinical symptoms are usually non-specific, new diagnostic and prognostic markers are urgently needed to enhance the number of identified cases and optimise treatment. This is particularly important for disease caused by Trypanosoma brucei rhodesiense, where indirect immunodiagnostic approaches have to date been unsuccessful. We have conducted global metabolic profiling of plasma from T.b.rhodesiense HAT patients and endemic controls, using 1H nuclear magnetic resonance (NMR) spectroscopy and ultra-performance liquid chromatography, coupled with mass spectrometry (UPLC-MS) and identified differences in the lipid, amino acid and metabolite profiles. Altogether 16 significantly disease discriminatory metabolite markers were found using NMR, and a further 37 lipid markers via UPLC-MS. These included significantly higher levels of phenylalanine, formate, creatinine, N-acetylated glycoprotein and triglycerides in patients relative to controls. HAT patients also displayed lower concentrations of histidine, sphingomyelins, lysophosphatidylcholines, and several polyunsaturated phosphatidylcholines. While the disease metabolite profile was partially consistent with previous data published in experimental rodent infection, we also found unique lipid and amino acid profile markers highlighting subtle but important differences between the host response to trypanosome infections between animal models and natural human infections. Our results demonstrate the potential of metabolic profiling in the identification of novel diagnostic biomarkers and the elucidation of pathogenetic mechanisms in this disease.
Anwar MA, Vorkas PA, Li JV, et al., 2015, Optimization of metabolite extraction of human vein tissue for ultra performance liquid chromatography-mass spectrometry and nuclear magnetic resonance-based untargeted metabolic profiling, Analyst, Vol: 140, Pages: 7586-7597, ISSN: 1364-5528
Human vein tissue is an important matrix to examine when investigating vascular diseases with respect to understanding underlying disease mechanisms. Here, we report the development of an extraction protocol for multi-platform metabolic profiling of human vein tissue. For the first stage of the optimization, two different ratios of methanol/water and 5 organic solvents – namely dichloromethane, chloroform, isopropanol, hexane and methyl tert-butyl ether (MTBE) solutions with methanol – were tested for polar and organic compound extraction, respectively. The extraction output was assessed using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and a panel of Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) methodologies. On the basis of the reproducibility of extraction replicates and metabolic coverage, the optimal aqueous (methanol/water) and organic (MTBE/methanol) solvents identified from the first stage were used in a sequential approach for metabolite extraction, altering the order of solvent-mixture addition. The combination of organic metabolite extraction with MTBE/methanol (3 : 1) followed by extraction of polar compounds with methanol/water (1 : 1) was shown to be the best method for extracting metabolites from human vein tissue in terms of reproducibility and number of signals detected and could be used as a single extraction procedure to serve both NMR and UPLC-MS analyses. Molecular classes such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines, sphingolipids, purines, and pyrimidines were reproducibly extracted. This study enabled an optimal extraction protocol for robust and more comprehensive metabolome coverage for human vein tissue. Many of the physiological and pathological processes affecting the composition of human vein tissue are common to other tissues and hence the extraction method developed in this study can be generically applied.
Vorkas PA, Isaac G, Holmgren A, et al., 2015, Perturbations in fatty acid metabolism and apoptosis are manifested in calcific coronary artery disease: An exploratory lipidomics study, International Journal of Cardiology, Vol: 197, Pages: 192-199, ISSN: 1874-1754
BackgroundControversy exists concerning the beneficial or harmful effects of the presence of ectopic calcification in the coronary arteries. Additionally, further elucidation of the exact pathophysiological mechanism is needed. In this study, we sought to identify metabolic markers of vascular calcification that could assist in understanding the disease, monitoring its progress and generating hypotheses describing its pathophysiology.MethodsUntargeted lipid profiling and complementary modeling strategies were employed to compare serum samples from patients with different levels of calcific coronary artery disease (CCAD) based on their calcium score (CS). Subsequently, patients were divided into three groups: no calcification (NC; CS = 0; n = 26), mild calcification (MC; CS:1–250; n = 27) and severe (SC; CS > 250; n = 17).ResultsPhosphatidylcholine levels were found to be significantly altered in the disease states (p = 0.001–0.04). Specifically, 18-carbon fatty acyl chain (FAC) phosphatidylcholines were detected in lower levels in the SC group, while 20:4 FAC lipid species were detected in higher concentrations. A statistical trend was observed with phosphatidylcholine lipids in the MC group, showing the same tendency as with the SC group. We also observed several sphingomyelin signals present at lower intensities in SC when compared with NC or MC groups (p = 0.000001–0.01).ConclusionsThis is the first lipid profiling study reported in CCAD. Our data demonstrate dysregulations of phosphatidylcholine lipid species, which suggest perturbations in fatty acid elongation/desaturation. The altered levels of the 18-carbon and 20:4 FAC lipids may be indicative of disturbed inflammation homeostasis. The marked sphingomyelin dysregulation in SC is consistent with profound apoptosis as a potential mechanism of CCAD.
Qureshi MI, Vorkas P, Shalhoub J, et al., 2015, Biomarker research in thromboembolic stroke (bruits), Annual Meeting of the Society-of-Academic-and-Research-Surgery (SARS(, Publisher: WILEY-BLACKWELL, Pages: 25-25, ISSN: 0007-1323
Vorkas PA, Shalhoub J, Isaac G, et al., 2015, Metabolic phenotyping of atherosclerotic plaques reveals latent associations between free cholesterol and ceramide metabolism in atherogenesis., Journal of proteome research, Vol: 14, Pages: 1389-1399, ISSN: 1535-3893
Current optimum medical treatments have had limited success in the primary prevention of cardiovascular events, underscoring the need for new pharmaceutical targets and enhanced understanding of mechanistic metabolic dysregulation. Here, we use a combination of novel metabolic profiling methodologies, based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) followed by chemometric modeling, data integration, and pathway mapping, to create a systems-level metabolic atlas of atherogenesis. We apply this workflow to compare arterial tissue incorporating plaque lesions to intimal thickening tissue (immediate preplaque stage). We find changes in several metabolite species consistent with well-established pathways in atherosclerosis, such as the cholesterol, purine, pyrimidine, and ceramide pathways. We then illustrate differential levels of previously unassociated lipids to atherogenesis, namely, phosphatidylethanolamine-ceramides (t-test p-values: 3.8 × 10(-6) to 9.8 × 10(-12)). Most importantly, these molecules appear to be interfacing two pathways recognized for their involvement in atherosclerosis: ceramide and cholesterol. Furthermore, we show that β-oxidation intermediates (i.e., acylcarnitines) manifest a pattern indicating truncation of the process and overall dysregulation of fatty acid metabolism and mitochondrial dysfunction. We develop a metabolic framework that offers the ability to map significant statistical associations between detected biomarkers. These dysregulated molecules and consequent pathway modulations may provide novel targets for pharmacotherapeutic intervention.
Vorkas PA, Isaac G, Anwar MA, et al., 2015, Untargeted UPLC-MS Profiling Pipeline to Expand Tissue Metabolome Coverage: Application to Cardiovascular Disease., Analytical Chemistry, Vol: 87, Pages: 4184-4193, ISSN: 1086-4377
Metabolic profiling studies aim to achieve broad metabolome coverage in specific biological samples. However, wide metabolome coverage has proven difficult to achieve, mostly because of the diverse physicochemical properties of small molecules, obligating analysts to seek multiplatform and multimethod approaches. Challenges are even greater when it comes to applications to tissue samples, where tissue lysis and metabolite extraction can induce significant systematic variation in composition. We have developed a pipeline for obtaining the aqueous and organic compounds from diseased arterial tissue using two consecutive extractions, followed by a different untargeted UPLC-MS analysis method for each extract. Methods were rationally chosen and optimized to address the different physicochemical properties of each extract: hydrophilic interaction liquid chromatography (HILIC) for the aqueous extract and reversed-phase chromatography for the organic. This pipeline can be generic for tissue analysis as demonstrated by applications to different tissue types. The experimental setup and fast turnaround time of the two methods contributed toward obtaining highly reproducible features with exceptional chromatographic performance (CV % < 0.5%), making this pipeline suitable for metabolic profiling applications. We structurally assigned 226 metabolites from a range of chemical classes (e.g., carnitines, α-amino acids, purines, pyrimidines, phospholipids, sphingolipids, free fatty acids, and glycerolipids) which were mapped to their corresponding pathways, biological functions and known disease mechanisms. The combination of the two untargeted UPLC-MS methods showed high metabolite complementarity. We demonstrate the application of this pipeline to cardiovascular disease, where we show that the analyzed diseased groups (n = 120) of arterial tissue could be distinguished based on their metabolic profiles.
Mirnezami R, Spagou K, Vorkas PA, et al., 2014, Chemical mapping of the colorectal cancer microenvironment via MALDI imaging mass spectrometry (MALDI-MSI) reveals novel cancer-associated field effects, Molecular Oncology, Vol: 8, Pages: 39-49, ISSN: 1574-7891
Matrix‐assisted laser desorption ionisation imaging mass spectrometry (MALDI‐MSI) is a rapidly advancing technique for intact tissue analysis that allows simultaneous localisation and quantification of biomolecules in different histological regions of interest. This approach can potentially offer novel insights into tumour microenvironmental (TME) biochemistry. In this study we employed MALDI‐MSI to evaluate fresh frozen sections of colorectal cancer (CRC) tissue and adjacent healthy mucosa obtained from 12 consenting patients undergoing surgery for confirmed CRC. Specifically, we sought to address three objectives: (1) To identify biochemical differences between different morphological regions within the CRC TME; (2) To characterise the biochemical differences between cancerous and healthy colorectal tissue using MALDI‐MSI; (3) To determine whether MALDI‐MSI profiling of tumour‐adjacent tissue can identify novel metabolic ‘field effects’ associated with cancer. Our results demonstrate that CRC tissue harbours characteristic phospholipid signatures compared with healthy tissue and additionally, different tissue regions within the CRC TME reveal distinct biochemical profiles. Furthermore we observed biochemical differences between tumour‐adjacent and tumour‐remote healthy mucosa. We have referred to this ‘field effect’, exhibited by the tumour locale, as cancer‐adjacent metaboplasia (CAM) and this finding builds on the established concept of field cancerisation.
Anwar MA, Vorkas P, Li J, et al., 2014, Differential Metabolic Phenotype of Human Varicose Veins Tissue and Their Utility in Understanding Disease Pathogenesis and Identifying Potential Prognostic Biomarkers., J Vasc Surg Venous Lymphat Disord, Vol: 2, Pages: 113-113, ISSN: 2213-333X
Markou A, Sourvinou I, Vorkas PA, et al., 2013, Clinical evaluation of microRNA expression profiling in non small cell lung cancer, LUNG CANCER, Vol: 81, Pages: 388-396, ISSN: 0169-5002
Anwar MA, Vorkas P, Li J, et al., 2013, Prolonged Mechanical Stretch Alters the Metabolic Profile in Rat Inferior Vena Cava., J Vasc Surg Venous Lymphat Disord, Vol: 1, Pages: 105-106, ISSN: 2213-333X
Anwar MA, Shalhoub J, Vorkas PA, et al., 2012, In-vitro Identification of Distinctive Metabolic Signatures of Intact Varicose Vein Tissue via Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy, EUROPEAN JOURNAL OF VASCULAR AND ENDOVASCULAR SURGERY, Vol: 44, Pages: 442-450, ISSN: 1078-5884
Mirnezami R, Kinross JM, Vorkas PA, et al., 2012, Implementation of Molecular Phenotyping Approaches in the Personalized Surgical Patient Journey, ANNALS OF SURGERY, Vol: 255, Pages: 881-889, ISSN: 0003-4932
Schiza C, Farkona S, Chimonidou M, et al., 2012, Detection of PIK3CA somatic mutations in cell-free DNA of breast cancer patients by high-resolution melting curve analysis, Publisher: AMER ASSOC CANCER RESEARCH, ISSN: 0008-5472
Dimitrakopoulos L, Vorkas PA, Georgoulias V, et al., 2012, A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples, BMC Cancer, Vol: 12, Pages: 486-486, ISSN: 1471-2407
BackgroundCST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.MethodsWe designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.ResultsThe developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.ConclusionsThe developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.
Vorkas PA, Poumpouridou N, Agelaki S, et al., 2010, PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method, JOURNAL OF MOLECULAR DIAGNOSTICS, Vol: 12, Pages: 697-704, ISSN: 1525-1578
Vorkas PA, Christopoulos K, Kroupis C, et al., 2010, Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis, Clinical Biochemistry, Vol: 43, Pages: 178-185, ISSN: 0009-9120
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