Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis

Devonshire, Alison S.; Sanders, Rebecca; Wilkes, Timothy; Taylor, Martin S.; Foy, Carole A.; Huggett, Jim F.

doi:10.1016/j.ymeth.2012.07.021

Cited by 60 publications

(40 citation statements)

References 112 publications

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“…The advent of next generation high throughput qPCR, based on reaction volumes scaled to the nanoliter range and with a consequent dramatic reduction in the volume of reagents and samples, has been a major advance for the analysis of clinical samples [13]. The Fluidigm Biomark system, one of the new high-throughput reverse transcription PCR (HT RT-qPCR) systems, permits up to 96 transcripts in 96 samples to be studied simultaneously during a single run, in a total of 9216 reactions.…”

Section: Introductionmentioning

confidence: 99%

A Method for Quantitative Analysis of Standard and High-Throughput qPCR Expression Data Based on Input Sample Quantity

2014

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Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR) have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR) and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells) and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA)) permits the normalization of results between different batches and between different instruments – regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1) the achievement of absolute quantification and (2) a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available.

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Section: Introductionmentioning

confidence: 99%

A Method for Quantitative Analysis of Standard and High-Throughput qPCR Expression Data Based on Input Sample Quantity

2014

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“…For example, many methods have been developed for the determination of mRNA levels in biological samples, based on separation by gel electrophoresis (GE) and detection by probe hybridization (northern analysis), PCR ampli cation of target sequence and GE, real-time PCR, microarray using hybridization to antisense probes, and RNaseq using the read count by next generation sequencer. 96) Digital PCR is a new technique for direct counting of the copy number of mRNA in samples without using calibration curves. 97) Transcriptomics research has been driven by the development of new technologies.…”

Section: Rethinking Instrumentation Of Mass Spectrometry For Metabolomentioning

confidence: 99%

Technical Challenges in Mass Spectrometry-Based Metabolomics

Mizutani

2016

Mass Spectrometry

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Metabolomics is a strategy for analysis, and quanti cation of the complete collection of metabolites present in biological samples. Metabolomics is an emerging area of scienti c research because there are many application areas including clinical, agricultural, and medical researches for the biomarker discovery and the metabolic system analysis by employing widely targeted analysis of a few hundred preselected metabolites from 10-100 biological samples. Further improvement in technologies of mass spectrometry in terms of experimental design for larger scale analysis, computational methods for tandem mass spectrometry-based elucidation of metabolites, and speci c instrumentation for advanced bioanalysis will enable more comprehensive metabolome analysis for exploring the hidden secrets of metabolism.

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“…These methods, known as high-throughput RT-qPCR (HT RTqPCR) or nanofluidic qPCR, permit the rapid quantification of multiple transcripts using small sample volumes [65,66] with very high sensitivity. Plates can contain up to 96 samples in which 96 transcripts can be simultaneously studied in 9216 reactions.…”

Section: Specific Assay Issuesmentioning

confidence: 99%

Recent and near-future advances in nucleic acid-based diagnosis of stroke

Baird

Soper

Pullagurla

et al. 2015

Expert Review of Molecular Diagnostics

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Stroke is a leading cause of death and disability in adults, but at present, treatment for ischemic stroke reaches only a small percentage of patients. This is because of the very short time window for treatment and the time-consuming evaluation involved. Intense efforts are underway to find novel approaches to expedite stroke diagnosis and treatment. In this review, we provide the rationale for the use of blood-based nucleic acid biomarkers to advance stroke diagnosis. We describe mRNA markers identified in genomic profiling of circulating leukocytes and then outline technological issues involved in the application of these results. We then describe the novel point-of-care technology that is in development for the rapid detection of multiple mRNA molecules in circulating leukocytes.

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Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis

Cited by 60 publications

References 112 publications

A Method for Quantitative Analysis of Standard and High-Throughput qPCR Expression Data Based on Input Sample Quantity

A Method for Quantitative Analysis of Standard and High-Throughput qPCR Expression Data Based on Input Sample Quantity

Technical Challenges in Mass Spectrometry-Based Metabolomics

Recent and near-future advances in nucleic acid-based diagnosis of stroke

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