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Detection of antisense RNA transcripts by strand-specific RT-PCR.
Eric C H Ho1, Michael E Donaldson, Barry J Saville
1Department of Medical Biophysics, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, Canada.
Methods in Molecular Biology (Clifton, N.J.)
|March 20, 2010
Summary
This study details a reverse transcription polymerase chain reaction (RT-PCR) method for detecting natural antisense transcripts (NATs). The optimized protocol efficiently screens for these regulatory RNA molecules, crucial for understanding cellular processes.
Area of Science:
- Molecular Biology
- Genomics
- RNA Biology
Background:
- Comprehensive genome annotation relies on extensive cDNA analysis.
- Natural antisense transcripts (NATs) have been identified across diverse eukaryotes, suggesting a conserved regulatory role.
- Confirming NATs and their functions requires robust detection methods.
Purpose of the Study:
- To describe an optimized reverse transcription polymerase chain reaction (RT-PCR) method for detecting natural antisense transcripts (NATs).
- To streamline the screening process for antisense transcripts by minimizing first-strand synthesis reactions.
- To provide a reliable method for confirming expressed sequence tag (EST) data and detecting NATs in various cellular contexts.
Main Methods:
- Utilized a reverse transcription polymerase chain reaction (RT-PCR) approach for NAT detection.
- Employed antisense-directed primers and oligo dT to prime first-strand cDNA synthesis, reducing the number of reactions.
- Confirmed results using both sense and antisense-directed primers for first-strand synthesis.
- Incorporated controls to eliminate genomic DNA (gDNA) contamination and rule out self-priming artifacts.
Main Results:
- Developed an efficient RT-PCR protocol for the detection of natural antisense transcripts (NATs).
- The optimized method reduces the number of necessary first-strand synthesis reactions.
- Demonstrated the importance of rigorous controls for gDNA contamination and self-priming in accurate NAT detection.
Conclusions:
- The described RT-PCR protocol offers an effective means for identifying and confirming natural antisense transcripts (NATs).
- This method facilitates further investigation into the significant cellular roles of NATs.
- Optimization and proper controls are essential for reliable detection of antisense transcripts.
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