Massively Parallel Reporter Assay
Massively Parallel Reporter Assay - Related Videos
11:03
Massively Parallel Reporter Assays in Cultured Mammalian Cells
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Cited by 63 •
2014
The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.
11:02
Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing
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Cited by 64 •
2013
We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.
10:45
A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules
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Cited by 2 •
2020
The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.
06:38
Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing
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Cited by 69 •
2018
Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.
02:01
Assessing Pseudovirus Infection Using Luciferase Reporter Assay
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2026
Source: Chang, X., et al. Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity. J. Vis. Exp. (2021).This video demonstrates a luciferase-based assay to quantify pseudovirus infection in epithelial cells. The pseudovirus encodes the luciferase gene, which is expressed in infected cells. After washing, and cells are incubated with lysis buffer at an ultra-low temperature, and then thawed to induce lysis.
FAQs
Sequencing the two barcode forms separates reporter output from construct representation. Barcode RNA reflects transcripts produced after a regulatory sequence acts, whereas barcode DNA indicates how much of the corresponding construct is present in the assay. Comparing these measurements helps quantify activity relative to representation, so differences are less likely to reflect unequal construct abundance alone.
A unique barcode creates a compact identifier for each tested sequence, allowing many constructs to be analyzed together by sequencing. The barcode connects a sequence’s identity to its RNA and DNA counts. This multiplexed design supports comparisons among thousands of candidate elements, genetic variants, or synthetic sequences within the same experimental framework.
MPRA can test how candidate enhancers, promoters, transcription-factor binding sites, and synthetic regulatory sequences influence reporter expression. By comparing barcode-based activity across designed sequences or genetic variants, investigators can identify sequence features associated with stronger or weaker transcription. These measurements provide a systematic way to examine regulatory logic within noncoding DNA.
An experiment begins by cloning selected regulatory sequences upstream of a reporter gene and assigning each construct a unique DNA barcode. The constructs are introduced into cells, after which researchers sequence barcode DNA and barcode RNA. They then compare RNA output with DNA representation to estimate the regulatory activity associated with each sequence.
Researchers can compare reporter activity for alternative versions of a sequence to determine whether a genetic variant changes regulatory output. When variants produce different activity measurements, the results identify possible effects on transcriptional control. These comparisons help connect noncoding mutations with biological traits or disease and prioritize regulatory sequences for further investigation.
Because many elements are measured simultaneously, the assay can reveal patterns across regulatory sequences rather than focusing on one candidate at a time. Activity measurements can show which sequence features correspond to transcriptional differences and support systematic analysis of noncoding genome function. This makes the approach useful for studying natural variants and engineered regulatory sequences in biology.