In Situ mRNA Detection via cDNA Padlock Probe Rolling Circle Amplification
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Solution Overview
Problem
Current methods for in situ detection of RNA, particularly mRNA, in formalin-fixed paraffin-embedded (FFPE) samples face challenges such as low detection efficiency and specificity due to RNA secondary structures and the need for specific site positioning, limiting the ability to study single nucleotide variations and allelic expression.
Innovation Solution
The method involves converting RNA to complementary DNA (cDNA) and using padlock probes for targeted rolling circle amplification (RCA), allowing for localized detection of RNA by hybridizing padlock probes to cDNA, which are then circularized and amplified, enabling sensitive and specific detection of mRNA and its variations in FFPE samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If direct RNA detection with padlock probes is performed in situ, then localization information is preserved, but detection efficiency and specificity are low due to RNA secondary structures and ligation inefficiency
Solution Approach 1:
The patent introduces cDNA as an intermediary molecule between RNA and padlock probe detection. RNA is first reverse transcribed into cDNA, which then serves as the target for padlock probe hybridization and ligation. This intermediary step eliminates the problems of RNA secondary structures and low ligation efficiency, as cDNA is more stable and forms better hybridization with padlock probes, thereby significantly improving detection efficiency and specificity while preserving spatial localization information
Solution Approach 2:
The patent changes the molecular state parameter from RNA to cDNA through reverse transcription. This parameter change transforms the target molecule from a single-stranded, structurally complex RNA into a double-stranded, more stable cDNA format that is more amenable to padlock probe ligation, thus resolving the technical contradiction between maintaining localization and improving detection reliability
2Measurement precision
If FISH is used to detect single mRNA molecules in situ, then transcript copy numbers can be determined, but highly similar sequences cannot be resolved
Solution Approach 1:
The patent segments the detection process into distinct molecular steps: reverse transcription of RNA to cDNA, hybridization of padlock probes to cDNA, ligation of padlock probes, and rolling circle amplification. Each segment is optimized for specific purposes, with padlock probes providing sequence-specific recognition that can distinguish highly similar sequences while rolling circle amplification providing the sensitivity needed for single-molecule detection, thus resolving the contradiction between measurement precision and detection sensitivity
3Measurement precision
If PCR of laser-capture microdissected material is performed to assign transcript variants to single cells, then sequence information can be obtained, but the process is time consuming and error prone
Solution Approach 1:
The patent employs rolling circle amplification that is self-priming and automatically amplifies the padlock probe-cDNA hybrid complexes in situ. The amplified rolling circle products serve as their own detection signals, eliminating the need for separate PCR amplification steps and complex laser-capture microdissection procedures. This self-service amplification mechanism maintains sequence accuracy while dramatically reducing detection time and operational complexity
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 the detection of single nucleotide variations and allelic expression in FFPE samples with high sensitivity and specificity, allowing for the study of transcriptional variations at the single-cell level, which is not achievable with existing methods.
Implementation Method 1
The method relies on the conversion of RNA to complementary DNA (cDNA) prior to the targeting of the cDNA with a padlock probe(s)
Implementation Method 2
The hybridization of the padlock probe(s) relies on the nucleotide sequence of the cDNA which is derived from the corresponding nucleotide sequence of the target RNA
Implementation Method 3
The circularized padlock probe is then subjected to RCA
Implementation Method 4
Rolling circle amplification (RCA) of the subsequently circularized padlock probe produces a rolling circle product (RCP)
Data Source
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AI summary
The present invention relates to the detection of RNA in a sample of cells. More particularly, the present invention relates to the localized detection of RNA in situ. The method relies on the conversion of RNA to complementary DNA prior to the targeting of the cDNA with a padlock probe(s). The hybridization of the padlock probe(s) relies on the nucleotide sequence of the cDNA which is derived from the corresponding nucleotide sequence of the target RNA. Rolling circle amplification of the subsequently circularized padlock probe produces a rolling circle product which may be detected. Advantageously, this allows the RNA to be detected in situ.