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Screening for ribosomal-based false positives following prokaryotic mRNA differential display
A C Nagel1, J T Fleming, G S Sayler
1University of Tennessee Knoxville, USA
Biotechniques
|May 18, 2001
Summary
Differential display (DD) and RNA arbitrarily primed PCR (RAP-PCR) identify gene expression changes but often yield false positives. This study uses ribosomal DNA (rDNA) probes to accurately distinguish true mRNA differences from abundant rRNA in prokaryotic DD analysis.
Area of Science:
- Molecular Biology
- Genomics
- Microbial Genetics
Background:
- Differential display (DD) and RNA arbitrarily primed PCR (RAP-PCR) are key techniques for identifying differentially expressed genes.
- A significant limitation of these methods, particularly in prokaryotes, is the generation of numerous false positives, often due to highly abundant ribosomal RNA (rRNA).
Purpose of the Study:
- To develop a prescreening method to reduce false positives in prokaryotic mRNA differential display.
- To accurately differentiate between differentially expressed messenger RNAs (mRNAs) and abundant ribosomal RNAs (rRNAs) in DD analysis.
Main Methods:
- Utilized full-length 16S and 23S ribosomal DNA (rDNA) probes derived from specific bacterial strains (Pseudomonas putida G7 and Pseudomonas aeruginosa FRD1).
- Applied these rDNA probes as a prescreening step in the DD workflow for prokaryotic mRNA analysis.
Main Results:
- The prescreening approach effectively discriminated between rRNA bands and potential mRNA candidates.
- This method successfully reduced the number of false positives typically encountered in prokaryotic DD experiments.
Conclusions:
- Employing full-length 16S and 23S rDNA probes is an effective strategy to eliminate rRNA as a source of false positives in prokaryotic DD.
- This prescreening method enhances the accuracy and reliability of identifying differentially expressed genes using DD in prokaryotic systems.