Stabilizing Extracellular Nucleic Acids Without Cell Lysis
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Solution Overview
Problem
Existing methods for stabilizing cell-containing samples, such as blood, often result in cell lysis, which contaminates extracellular nucleic acids with intracellular nucleic acids, making it difficult to isolate and analyze these molecules accurately.
Innovation Solution
The use of N,N-dialkylpropanamides, such as N,N-dimethylpropanamide, in combination with apoptosis inhibitors and chelating agents, which contact with the cell-containing sample to stabilize both the extracellular nucleic acid population and the gene expression profile of contained cells without causing cell lysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional stabilisation methods (organic solvents, direct freezing) are used, then RNA stability is improved, but cell lysis occurs causing contamination of extracellular nucleic acids with intracellular nucleic acids
Solution Approach 1:
The patent changes the chemical parameters of the stabilisation system by using N,N-dialkylpropanamides (specific esters) instead of conventional organic solvents like phenol or chloroform. This parameter change allows stabilisation at physiological pH and temperature without causing cell lysis, thereby preventing contamination while maintaining RNA stability.
Solution Approach 2:
The patent introduces N,N-dialkylpropanamides as intermediary substances that mediate between the need for RNA stabilisation and the requirement to preserve cell integrity. These esters act as RNAse inhibitors that stabilise extracellular nucleic acids without the harsh effects of conventional methods, serving as a bridge solution that satisfies both conflicting requirements.
2Reliability
If immediate lysis methods are used for stabilisation, then RNA degradation is prevented, but separate analysis of extracellular and intracellular nucleic acids becomes impossible
Solution Approach 1:
The patent applies preliminary stabilisation action using N,N-dialkylpropanamides that prevent RNA degradation in extracellular space while maintaining cell integrity. This preliminary protection allows subsequent separate analysis of extracellular and intracellular nucleic acids, as the extracellular RNA is already stabilised before any lysis occurs.
Solution Approach 2:
The patent enables segmentation of nucleic acid analysis by stabilising extracellular RNA separately from intracellular RNA. The N,N-dialkylpropanamide treatment creates distinct stabilised extracellular nucleic acid populations that can be separately isolated and analysed from intracellular contents, providing analytical segmentation.
3Ease of operation
If prolonged storage at room temperature is required, then transport convenience is improved, but gene expression profiles become altered
Solution Approach 1:
The patent ensures continuous protection of gene expression profiles during prolonged room temperature storage by maintaining persistent RNAse inhibition through N,N-dialkylpropanamide treatment. This continuous protective action allows convenient transport without refrigeration while preserving the original gene expression profile throughout the storage period.
Solution Approach 2:
The patent applies beforehand cushioning by pre-treating samples with N,N-dialkylpropanamides that provide advance protection against RNA degradation and gene expression changes during subsequent prolonged storage. This prior protection cushions the sample against the harmful effects of room temperature storage, allowing transport convenience without profile alteration.
Data Source
AI summary
The present invention provides methods, compositions and devices for stabilizing the extracellular nucleic acid population in a cell-containing biological sample and for stabilizing the transcriptome of contained cells.