Evaluation of a novel approach for the measurement of RNA quality

<p>Abstract</p> <p>Background</p> <p>Microarray data interpretation can be affected by sample RNA integrity. The ScreenTape Degradation Value (SDV) is a novel RNA integrity metric specific to the ScreenTape^®platform (Lab901). To characterise the performance of the ScreenTape^®platform for RNA analysis and determine the robustness of the SDV metric, a panel of intentionally degraded RNA samples was prepared. These samples were used to evaluate the ScreenTape^®platform against an alternative approach for measuring RNA integrity (Agilent Bioanalyzer RIN value). The samples were also subjected to microarray analysis and the resulting data correlated to the RNA integrity metrics.</p> <p>Findings</p> <p>Measurement of SDV for a panel of intentionally degraded RNA samples ranged from 0 for intact RNA to 37 for degraded RNA, with corresponding RIN values ranging from 10 to 4 for the same set of samples. SDV and RIN scales both demonstrated comparable discrimination between differently treated samples (RIN 10 to 7, SDV 0 to 15), with the SDV exhibiting better discrimination at higher degradation levels. Increasing SDV values correlated with a decrease in microarray sample labelling efficiency and an increase in numbers of differentially expressed genes.</p> <p>Conclusions</p> <p>The ScreenTape^®platform is comparable to the Bioanalyzer platform in terms of reproducibility and discrimination between different levels of RNA degradation. The robust nature of the SDV metric qualifies it as an alternative metric for RNA sample quality control, and a useful predictor of downstream microarray performance.</p

Quantification of epigenetic biomarkers:an evaluation of established and emerging methods for DNA methylation analysis

BACKGROUND: DNA methylation is an important epigenetic mechanism in several human diseases, most notably cancer. The quantitative analysis of DNA methylation patterns has the potential to serve as diagnostic and prognostic biomarkers, however, there is currently a lack of consensus regarding the optimal methodologies to quantify methylation status. To address this issue we compared five analytical methods: (i) MethyLight qPCR, (ii) MethyLight digital PCR (dPCR), methylation-sensitive and -dependent restriction enzyme (MSRE/MDRE) digestion followed by (iii) qPCR or (iv) dPCR, and (v) bisulfite amplicon next generation sequencing (NGS). The techniques were evaluated for linearity, accuracy and precision. RESULTS: MethyLight qPCR displayed the best linearity across the range of tested samples. Observed methylation measured by MethyLight- and MSRE/MDRE-qPCR and -dPCR were not significantly different to expected values whilst bisulfite amplicon NGS analysis over-estimated methylation content. Bisulfite amplicon NGS showed good precision, whilst the lower precision of qPCR and dPCR analysis precluded discrimination of differences of < 25% in methylation status. A novel dPCR MethyLight assay is also described as a potential method for absolute quantification that simultaneously measures both sense and antisense DNA strands following bisulfite treatment. CONCLUSIONS: Our findings comprise a comprehensive benchmark for the quantitative accuracy of key methods for methylation analysis and demonstrate their applicability to the quantification of circulating tumour DNA biomarkers by using sample concentrations that are representative of typical clinical isolates. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-1174) contains supplementary material, which is available to authorized users

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine

Applicability of RNA standards for evaluating RT-qPCR assays and platforms

The availability of diverse RT-qPCR assay formats and technologies hinder comparability of data between platforms. Reference standards to facilitate platform evaluation and comparability are needed. We have explored using universal RNA standards for comparing the performance of a novel qPCR platform (Fluidigm® BioMark™) against the widely used ABI 7900HT system. Our results show that such standards may form part of a toolkit to evaluate the key performance characteristics of platforms

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology

The role of hepatocyte nuclear factor 4α (HNF4α) in the metabolic regulation of its target genes

The nuclear receptor Hepatocyte Nuclear Factor 4α (HNF4α; NR2A1) regulates the transcription of many genes involved in glucose and lipid metabolism. Genetic linkage analyses have implicated HNF4α in the disease processes leading to Type 2 Diabetes Mellitus and dyslipidaemia. The aim of this study was to investigate the regulation of tf target genes in the metabolic pathways of glycolysis, lipogenesis and gluconeogenesis by HNF4α.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The role of hepatocyte nuclear factor 4alpha (HNF4alpha) in the metabolic regulation of its target genes.

The nuclear receptor Hepatocyte Nuclear Factor 4a (HNF4a; NR2A1) regulates the transcription of many genes involved in glucose and lipid metabolism. Genetic linkage analyses have implicated HNF4a in the disease processes leading to Type 2 Diabetes Mellitus and dyslipidaemia. The aim of this study was to investigate the regulation of target genes in the metabolic pathways of glycolysis, lipogenesis and gluconeogenesis by HNF4a. Initally, the expression of HNF4a and its splice variants was investigated in three human hepatoma cell lines, HuH7, HepG2 and Hep3B, with the latter two cell lines shown to express the same range of HNF4a splice variants as human adult liver. The regulation of specific HNF4a target genes, L-PK, PEPCK and SREBP-1c, was subsequently investigated in HepG2 cells using a reporter gene approach. HNF4a was found to induce expression of reporter genes containing L-PK, PEPCK and SREBP-1c proximal promoter sequences. Insulin (1 ?M), but not high glucose (25 mM), was found to stimulate HNF4a-driven expression of the SREBP-1c reporter gene, while co-expression of HNF4a with the nuclear receptor coactivators, PGC-1a or p300, led to a reduction in SREBP-1c reporter gene expression. The changes in expression of various HNF4a target genes in response to physiological mediators of the fasting-fed cycle were characterised in HepG2 cells using a real-time quantitative PCR approach. The role of HNF4a, p300 and PGC-1a was further investigated by plasmid overexpression. HNF4a and PGC-1a were found to positively regulate PEPCK expression under cell culture conditions simulating fasting (cAMP), whilst overexpression of HNF4a, PGC1a and p300 reduced L-PK mRNA expression under fed conditions. In conclusion, the results indicate that HNF4a is a transcriptional activator of both glucagon- stimulated gluconeogenic gene expression and insulin-stimulated glycolytic and lipogenic gene expression. It is hypothesised HNF4a forms separate multi-protein complexes to differentially regulate metabolic pathways under different metabolic states

The role of hepatocyte nuclear factor 4alpha (HNF4alpha) in the metabolic regulation of its target genes.

The nuclear receptor Hepatocyte Nuclear Factor 4a (HNF4a; NR2A1) regulates the transcription of many genes involved in glucose and lipid metabolism. Genetic linkage analyses have implicated HNF4a in the disease processes leading to Type 2 Diabetes Mellitus and dyslipidaemia. The aim of this study was to investigate the regulation of target genes in the metabolic pathways of glycolysis, lipogenesis and gluconeogenesis by HNF4a. Initally, the expression of HNF4a and its splice variants was investigated in three human hepatoma cell lines, HuH7, HepG2 and Hep3B, with the latter two cell lines shown to express the same range of HNF4a splice variants as human adult liver. The regulation of specific HNF4a target genes, L-PK, PEPCK and SREBP-1c, was subsequently investigated in HepG2 cells using a reporter gene approach. HNF4a was found to induce expression of reporter genes containing L-PK, PEPCK and SREBP-1c proximal promoter sequences. Insulin (1 ?M), but not high glucose (25 mM), was found to stimulate HNF4a-driven expression of the SREBP-1c reporter gene, while co-expression of HNF4a with the nuclear receptor coactivators, PGC-1a or p300, led to a reduction in SREBP-1c reporter gene expression. The changes in expression of various HNF4a target genes in response to physiological mediators of the fasting-fed cycle were characterised in HepG2 cells using a real-time quantitative PCR approach. The role of HNF4a, p300 and PGC-1a was further investigated by plasmid overexpression. HNF4a and PGC-1a were found to positively regulate PEPCK expression under cell culture conditions simulating fasting (cAMP), whilst overexpression of HNF4a, PGC1a and p300 reduced L-PK mRNA expression under fed conditions. In conclusion, the results indicate that HNF4a is a transcriptional activator of both glucagon- stimulated gluconeogenic gene expression and insulin-stimulated glycolytic and lipogenic gene expression. It is hypothesised HNF4a forms separate multi-protein complexes to differentially regulate metabolic pathways under different metabolic states