Molecular diagnostics assays use genetic or protein biomarkers to diagnose disease, monitor disease progression, assess drug efficacy, or stratify patients for clinical treatment.
In general, biomarker tools can be divided into two categories, discovery and testing. Tools in the former category identify biomarker patterns from large pools of potential biomarkers, while the latter applies the chosen marker to guide clinical care.
Nucleic acid biomarkers
Nucleic acid biomarkers include both genetic variants (eg SNPs or CNVs) and RNAs (especially fusion transcripts in cancer and aberrant microRNA expression profiles). Genetic variants are detected via DNA sequencing. In discovery mode, whole-genome or whole-exome sequencing strategies generally are used to identify differences between affected and control populations. Once those variants are identified, however, they can be tested for using a more restricted, targeted approach, such as targeted DNA sequencing or PCR.
Likewise, RNA biomarkers are discovered using transcriptome-sequencing strategies (eg, RNA-seq), but typically assayed using lower cost, higher throughput methods, such as qPCR, microarrays or fluorescence in situ hybridization.
Protein biomarkers
Protein biomarker discovery is generally accomplished using high-resolution, high-mass-accuracy mass spectrometry (eg, an Orbitrap or MALDI-TOF-TOF), though other strategies, including 2D protein gel electrophoresis and protein microarrays also may be used. Whatever the approach, the goal is the same: To compare protein presence and abundance between affected and unaffected sample populations.
Once a biomarker – which may include one or more proteins -- has been identified, it can be quantified using low-resolution quantitative mass spectrometry (eg, on a tandem quadrupole mass spectrometer), ELISA, or any other affinity-based assay, such as immunofluorescence. One common approach used multiplexed beads, such as Luminex xMAP beads, to quantify multiple proteins simultaneously in a single sample.
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