Multiplexed Transcriptional Activity Detection via RNA Sequencing
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Solution Overview
Problem
Current methods for investigating cellular pathways are expensive and cumbersome, relying on multi-well microplates and robots, necessitating the development of alternative approaches for multiplexed detection of transcriptional activity modulation by perturbation elements.
Innovation Solution
A method involving the introduction of delivery vectors into cell cultures, each containing a perturbation element DNA sequence linked to a promoter of interest and a constitutive promoter, allowing expression and detection of perturbation element RNA sequences to quantify modulation of transcriptional activity, using DNA constructs and kits that include oligonucleotide primers for sequencing and analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multi-well microplates and robots are used for pathway specific screens, then measurement precision and reliability are improved, but device complexity and cost increase
Solution Approach 1:
The patent uses RNA sequencing to create a digital copy and quantification of transcriptional activity levels. Instead of complex physical measurements with robots and plate readers, the system sequences RNA molecules to generate digital data that accurately represents promoter activity, thereby achieving high measurement precision through information copying rather than complex physical measurement systems
Solution Approach 2:
The patent replaces mechanical systems (robots, microplate handlers, plate readers) with a molecular biology-based sequencing system. The mechanical automation of sample handling and measurement is substituted by in vitro transcription and RNA sequencing processes that automatically quantify promoter activity through molecular amplification and sequencing, eliminating the need for complex mechanical equipment
2Measurement precision
If multi-well microplates and robots are used for pathway specific screens, then measurement precision is improved, but cost increases
Solution Approach 1:
The patent uses RNA sequencing to create a digital copy and quantification of transcriptional activity levels. Instead of complex physical measurements with robots and plate readers, the system sequences RNA molecules to generate digital data that accurately represents promoter activity, thereby achieving high measurement precision through information copying rather than complex physical measurement systems
Solution Approach 2:
The RNA sequencing approach serves multiple functions simultaneously: it quantifies promoter activity, identifies active promoters, and provides digital data for analysis all in a single experiment. This multi-functionality eliminates the need for separate measurement steps and expensive specialized equipment, reducing overall cost while maintaining precision
3Measurement precision
If conventional screening methods are used, then detection accuracy is maintained, but productivity decreases
Solution Approach 1:
The patent combines multiple detection functions into a single RNA sequencing experiment. Instead of separate steps for measuring promoter activity, the system performs in vitro transcription from genomic DNA followed by RNA sequencing, which simultaneously quantifies all active promoters in parallel, thereby achieving high throughput while maintaining accurate detection through molecular amplification
Solution Approach 2:
The patent performs preliminary amplification of the target (RNA synthesis from DNA templates) before detection. This preliminary action creates abundant copies of the transcriptional activity signals, enabling highly sensitive and accurate detection even at low initial concentrations, while the amplified signals can be processed in high throughput through sequencing
4Device complexity
If alternative methods without robots and microplates are developed, then device complexity and cost are reduced, but measurement precision may worsen
Solution Approach 1:
The patent uses RNA sequencing to create a digital copy and quantification of transcriptional activity levels. Instead of complex physical measurements with robots and plate readers, the system sequences RNA molecules to generate digital data that accurately represents promoter activity, thereby achieving high measurement precision through information copying rather than complex physical measurement systems
Solution Approach 2:
The patent replaces mechanical systems (robots, microplate handlers, plate readers) with a molecular biology-based sequencing system. The mechanical automation of sample handling and measurement is substituted by in vitro transcription and RNA sequencing processes that automatically quantify promoter activity through molecular amplification and sequencing, eliminating the need for complex mechanical equipment
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient, multiplexed, and quantitative detection of transcriptional activity modulation by perturbation elements, reducing costs and complexity compared to existing technologies, while allowing for high-throughput analysis of genetic perturbations on cellular pathways.
Implementation Method 1
The cell culture is allowed to express the plurality of delivery vectors. The expression forms a plurality of perturbation element identifying RNA sequences from the perturbation element identifying DNA sequences
Data Source
AI summary
Provided herein are methods directed to multiplexed detection of the modulation of transcriptional activity using perturbation elements.