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A method for preventing artifactual binding of cRNA probes to neurons caused by in situ hybridization
Abstract:
When in situ hybridization was used for the detection of mRNA for the beta-trace protein (beta-trace; prostaglandin-D-synthase) in sections of rat and porcine brains, unspecific binding reactions of sense and antisense probes to neurons were observed. The beta-trace fragment which served as a template for the synthesis of cRNA probes was blunt end-cloned in the vector pCR-Script SK (+). It was demonstrated that the unspecific signals were caused by artifactual binding of two portions of the cRNA which correspond to sequences of the multicloning site of this vector. These sequences are localized between the SrfI restriction site (or the insert) and the promoter for the T7 RNA polymerase. Thus, artifactual binding could be prevented using riboprobes synthesized by T3 RNA polymerase instead of T7 RNA polymerase. Because of the relatively weak transcription efficiency of T3 RNA polymerase, as compared with T7 RNA polymerase, a blocking procedure was established which allowed successful in situ hybridization with T7 RNA polymerase-synthesized probes. Blocking was performed using synthetic oligonucleotides deduced from the two sequences of the multicloning site which were found to be responsible for artifactual binding.
Insights
Unspecific binding in brain tissue during in situ hybridization was caused by vector sequences in RNA probes. This artifactual binding can be prevented by using T3 RNA polymerase probes or blocking T7 RNA polymerase probes with oligonucleotides.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- In situ hybridization is a key technique for detecting mRNA in tissue sections.
- Unspecific binding of RNA probes can lead to inaccurate results in molecular biology studies.
- Beta-trace protein (beta-trace), also known as prostaglandin-D-synthase, plays a role in the brain.
Purpose of the Study:
- To investigate the cause of unspecific binding observed during in situ hybridization for beta-trace mRNA in rat and porcine brains.
- To develop methods to prevent artifactual binding and improve the accuracy of in situ hybridization.
Main Methods:
- In situ hybridization using sense and antisense cRNA probes synthesized from a beta-trace fragment cloned in the pCR-Script SK (+) vector.
- Analysis of unspecific binding sites by identifying vector sequences responsible for artifactual hybridization.
- Synthesis of riboprobes using T3 RNA polymerase as an alternative to T7 RNA polymerase.
- Development of a blocking procedure using synthetic oligonucleotides to prevent unspecific binding.
Main Results:
- Unspecific binding of RNA probes to neurons was observed in rat and porcine brain sections.
- The artifactual signals were attributed to the binding of vector sequences present in the cRNA probes.
- Using riboprobes synthesized by T3 RNA polymerase prevented artifactual binding.
- A blocking procedure with synthetic oligonucleotides successfully prevented unspecific binding with T7 RNA polymerase-synthesized probes.
Conclusions:
- Artifactual binding in in situ hybridization can be caused by sequences within the cloning vector used for probe synthesis.
- The use of T3 RNA polymerase or a blocking strategy with synthetic oligonucleotides can effectively prevent these unspecific signals.
- These findings provide a method to improve the reliability of in situ hybridization for detecting specific mRNA targets in neural tissues.