Cellular Sample Nucleic Acid Detection via Nuclease Inhibitor Protection
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Solution Overview
Problem
Current methods for assessing RNA expression in individual cells are limited in their ability to detect target nucleic acids with high sensitivity and specificity, particularly in preserving RNA integrity and allowing for simultaneous detection of RNA and protein markers.
Innovation Solution
The development of a method involving fixation, permeabilization, and post-fixation of cellular samples, followed by contact with a nuclease inhibitor, allows for the detection of target nucleic acids using branched DNA amplification and simultaneous co-detection of protein-based markers, enabling sensitive multiplexed analysis of mRNA transcripts and protein markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods are used to assess RNA expression in individual cells, then the analysis can be performed, but the detection sensitivity and specificity for target nucleic acids are limited
Solution Approach 1:
The method applies fixation and permeabilization treatments before RNA detection to prepare cells in advance. The fixation step crosslinks cellular components to preserve RNA integrity, while permeabilization creates pathways for probe access. These preliminary actions enable subsequent sensitive and specific detection without compromising RNA quality during the assay process.
Solution Approach 2:
The invention introduces nuclease inhibitors as intermediary substances that protect RNA from degradation during the detection process. These inhibitors act as mediators between the RNA target and the detection system, preventing harmful nuclease activity while allowing specific probe binding. This intermediary protection enables both high RNA integrity preservation and sensitive detection.
2Measurement precision
If fixation and permeabilization are applied to preserve RNA integrity, then RNA detection sensitivity improves, but the ability to simultaneously detect protein markers may be compromised
Solution Approach 1:
The fixation and permeabilization protocol is designed to serve multiple functions simultaneously. The same treatment conditions that optimize RNA preservation and accessibility also maintain protein epitope integrity and accessibility. This multi-functional approach enables the detection system to assess both RNA and protein markers in the same cell sample without requiring separate preparation protocols.
Solution Approach 2:
The method optimizes fixation and permeabilization parameters (such as reagent concentrations, treatment times, and temperatures) to achieve a balance that simultaneously preserves RNA integrity and maintains protein detectability. By carefully adjusting these parameters, the protocol enables sensitive RNA detection while keeping protein markers accessible for immunodetection.
3Ease of operation
If standard detection protocols are used, then the process is simple, but the ability to detect low concentration targets accurately is limited
Solution Approach 1:
The invention employs nuclease inhibitors and optimized hybridization probes as intermediary elements that enhance signal generation from low concentration targets. These intermediaries protect rare RNA molecules from degradation and facilitate efficient probe binding, thereby improving detection accuracy without significantly complicating the overall protocol. The approach maintains operational simplicity while achieving sensitive detection through carefully selected intermediary substances.
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
This approach enables sensitive and accurate detection of RNA and protein markers at low concentrations, preserving RNA integrity and allowing for the identification of aberrant cells, such as cancer cells, with improved sensitivity and specificity.
Implementation Method 1
evaluating a cellular sample that has been contacted with a nuclease inhibitor for the presence of a target nucleic acid
Implementation Method 2
allows for the detection of target nucleic acids using branched DNA amplification
Data Source
AI summary
Methods of assaying cells of a cellular sample for the presence of a target nucleic acid are provided. Aspects of the methods include evaluating a cellular sample that has been contacted with a nuclease inhibitor for the presence of a target nucleic acid. Also provided are devices and kits that find use in practicing the methods described herein.