Gene validation

       High-throughput sequencing and quantitative protein mass spectrometry are commonly used to analyze EV contents. Following identification of differentially expressed genes by sequencing, expression can be validated qualitatively and quantitatively in EVs from particular tissues or cells relative to controls. Functional studies, such as gene knockout or knockdown, overexpression or rescue experiments, can then examine associations with physiological or pathological states.

1. Quantitative real-time PCR (qPCR)

       The source describes real-time PCR, introduced by ABI in 1996, as an automated quantitative nucleic-acid method with advantages in specificity and contamination control over conventional PCR. Common approaches include SYBR Green I dye and TaqMan probes. Applications include DNA and RNA quantification, SNP and genotype analysis and RNA variant analysis.

2. Digital PCR (ddPCR)

       Digital PCR partitions nucleic-acid samples into many independent nanoliter reactions, some containing target molecules and others without them. After amplification, TaqMan chemistry or dye-labelled probes detect the target sequence. The source describes absolute target counting and advantages in specificity, sensitivity, quantitative accuracy and resistance to interference.

3. Luciferase reporter (Luc)

       A luciferase reporter system detects firefly luciferase activity using luciferin as a substrate. Luciferase catalyzes oxidation of luciferin to oxyluciferin, producing bioluminescence that can be measured with a luminometer or liquid-scintillation counter. The source describes this sensitive system for measuring gene expression and investigating interactions between transcription factors and a target promoter.