Parallel Biomolecule Extraction from FFPE Tissue
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Solution Overview
Problem
Existing methods fail to simultaneously isolate and analyze nucleic acids and proteins from formalin-fixed, paraffin-embedded (FFPE) tissues due to crosslinking, which prevents parallel extraction from a single sample, limiting diagnostic and research capabilities.
Innovation Solution
A method involving the use of a protease-free buffer, detergent, and boiling followed by incubation above 60°C to dissolve crosslinking, allowing for the separation of biomolecules into soluble and insoluble fractions, enabling the separate isolation and analysis of proteins and nucleic acids from the same sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Shape
If formalin fixing is used to preserve tissue morphology, then morphology preservation is improved, but parallel extraction of nucleic acids and proteins deteriorates due to crosslinking
Solution Approach 1:
The extraction process is divided into two separate pathways: one for protein extraction and one for nucleic acid extraction. After dissolving crosslinks and separating into fractions, proteins are isolated from the soluble fraction while nucleic acids are isolated from the insoluble fraction, allowing parallel extraction without interference
Solution Approach 2:
A protease-free buffer system acts as an intermediary to dissolve formalin crosslinks and enable separation of biomolecules. The buffer contains detergents and chaotropic agents that facilitate the dissolution of crosslinked structures without degrading proteins or nucleic acids
2Manufacturing precision
If protease is used to purify nucleic acids by destroying proteins, then nucleic acid purification is improved, but protein isolation simultaneously deteriorates
Solution Approach 1:
Proteins are extracted and isolated from the soluble fraction before nucleic acid purification begins. This removes proteins that would otherwise interfere with nucleic acid isolation, eliminating the need for protease treatment while preserving protein samples for separate analysis
Solution Approach 2:
Proteins are removed from the sample by extraction and precipitation before nucleic acid isolation. This preliminary removal of proteins prevents interference with subsequent nucleic acid purification steps without requiring proteolytic digestion
3Adaptability or versatility
If sample material is divided into subsamples for separate purifications, then multiple biomolecule types can be isolated, but sample representativeness deteriorates
Solution Approach 1:
Multiple biomolecule extraction pathways are merged into a single unified protocol starting from one undivided sample. The method processes the entire sample through crosslink dissolution and fractionation, then directs different fractions to appropriate isolation pathways, ensuring all analytes originate from the same representative material
4Adaptability or versatility
If multiple separate purifications are performed on small samples, then various biomolecules can be obtained, but sample sufficiency deteriorates
Solution Approach 1:
The extraction method is designed to be universal and multi-functional, capable of isolating proteins, DNA, and RNA from a single sample through one integrated protocol. The method handles multiple biomolecule types simultaneously by directing different fractions to appropriate isolation pathways, maximizing utilization of limited sample material
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 the quantitative and sensitive isolation of both proteins and nucleic acids from FFPE tissues, facilitating accurate diagnostic and therapeutic applications by ensuring intact biomolecules can be compared and analyzed from the same starting material.
Implementation Method 1
a) a step of dissolving said crosslinking of the starting material
Implementation Method 2
boiling followed by incubation above 60°C to dissolve crosslinking
Implementation Method 3
the nucleic acids can be removed substantially by a simple mechanical removal step, for example by centrifugation or filtration
Implementation Method 4
the nucleic acids can be removed substantially by a simple mechanical removal step, for example by centrifugation or filtration
Data Source
AI summary
The present invention relates to a method of isolating/extracting in parallel various biomolecules, in particular nucleic acids and proteins, from the same fixed biological samples, to the quantification and analysis of the biomolecules isolated by the method of the invention, and to a kit for isolating/extracting in parallel various biomolecules from a fixed sample, to the use of said kit for diagnosing, prognosing, deciding the therapy of and monitoring the therapy of a disease.