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

Interlaboratory evaluation of quality control methods for circulating cell-free DNA extraction

: Analysis of circulating cell-free DNA (ccfDNA) isolated from liquid biopsies is rapidly being implemented into clinical practice. However, diagnostic accuracy is significantly impacted by sample quality and standardised approaches for assessing the quality of ccfDNA are not yet established. In this study we evaluated the application of nucleic acid "spike-in" control materials to aid quality control (QC) and standardisation of cfDNA isolation for use in in vitro diagnostic assays. We describe an approach for the design and characterisation of in-process QC materials, illustrating it with a spike-in material containing an exogenous Arabidopsis sequence and DNA fragments approximating to ccfDNA and genomic DNA lengths. Protocols for inclusion of the spike-in material in plasma ccfDNA extraction and quantification of its recovery by digital PCR (dPCR) were assessed for their suitability for process QC in an inter-laboratory study between five expert laboratories, using a range of blood collection devices and ccfDNA extraction methods. The results successfully demonstrated that spiking plasmid-derived material into plasma did not deleteriously interfere with endogenous ccfDNA recovery. The approach performed consistently across a range of commonly-used extraction protocols and was able to highlight differences in efficiency and variability between the methods, with the dPCR quantification assay performing with good repeatability (generally CV <5%). We conclude that initial findings demonstrate that this approach appears "fit for purpose" and spike-in recovery can be combined with other extraction QC metrics for monitoring the performance of a process over time, or in the context of external quality assessment. AVAILABILITY OF DATA AND MATERIALS: Processed data is available in Supplementary File 4. Raw data available upon request. Declaration of Competing Interest

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

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA

Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-017-0206-0) contains supplementary material, which is available to authorized users

The SunBEAm Birth Cohort: Protocol Design

BACKGROUND: Food allergy (FA) and atopic dermatitis (AD) are common conditions that often present in the first year of life. Identification of underlying mechanisms and environmental determinants of FA and AD is essential to develop and implement effective prevention and treatment strategies. Objectives: We sought to describe the design of the Systems Biology of Early Atopy (SunBEAm) birth cohort. METHODS: Funded by the National Institute of Allergy and Infectious Diseases (NIAID) and administered through the Consortium for Food Allergy Research (CoFAR), SunBEAm is a US population-based, multicenter birth cohort that enrolls pregnant mothers, fathers, and their newborns and follows them to 3 years. Questionnaire and biosampling strategies were developed to apply a systems biology approach to identify environmental, immunologic, and multiomic determinants of AD, FA, and other allergic outcomes. RESULTS: Enrollment is currently underway. On the basis of an estimated FA prevalence of 6%, the enrollment goal is 2500 infants. AD is defined on the basis of questionnaire and assessment, and FA is defined by an algorithm combining history and testing. Although any FA will be recorded, we focus on the diagnosis of egg, milk, and peanut at 5 months, adding wheat, soy, cashew, hazelnut, walnut, codfish, shrimp, and sesame starting at 12 months. Sampling includes blood, hair, stool, dust, water, tape strips, skin swabs, nasal secretions, nasal swabs, saliva, urine, functional aspects of the skin, and maternal breast milk and vaginal swabs. CONCLUSIONS: The SunBEAm birth cohort will provide a rich repository of data and specimens to interrogate mechanisms and determinants of early allergic outcomes, with an emphasis on FA, AD, and systems biology

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 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