Related Experiment Videos
Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches.
C Rosenow1, R M Saxena, M Durst
1Affymetrix Inc., 3380 Central Expressway, Santa Clara, CA 95051, USA. carsten_rosenow@affymetrix.com
Nucleic Acids Research
|November 20, 2001
Summary
This study developed novel labeling methods for prokaryotic gene expression analysis using high-density oligonucleotide arrays. Both methods accurately detect gene expression changes in Escherichia coli.
Area of Science:
- Microbiology
- Genomics
- Molecular Biology
Background:
- High-density oligonucleotide arrays are common for eukaryotic expression studies.
- Existing labeling protocols are unsuitable for prokaryotic cell analysis.
- A need exists for effective prokaryotic gene expression monitoring.
Purpose of the Study:
- To design a prokaryotic high-density oligonucleotide array for the complete Escherichia coli genome.
- To develop and compare mRNA enrichment and direct labeling protocols for prokaryotic gene expression analysis.
- To assess the reproducibility and concordance of different labeling methods.
Main Methods:
- Designed a prokaryotic high-density oligonucleotide array for Escherichia coli.
- Developed mRNA enrichment and direct labeling protocols.
- Synthesized cDNA and performed direct labeling.
- Utilized high-density oligonucleotide probe arrays for expression level determination.
- Compared expression results from direct labeling and cDNA synthesis.
Main Results:
- Approximately 50% of annotated Escherichia coli open reading frames are transcribed in rich LB medium.
- Both labeling methods demonstrated high concordance in replica experiments (95% and 99%).
- A 32% discordance was observed when comparing the two sample preparation methods.
- Both methods successfully detected relative gene expression changes in induced versus uninduced cells.
Conclusions:
- Developed and validated novel labeling protocols for prokaryotic gene expression studies using oligonucleotide arrays.
- Identified a significant portion of the Escherichia coli genome as transcribed under standard growth conditions.
- Highlighted the importance of sample preparation methods in gene expression analysis while confirming the utility of both developed protocols for detecting relative changes.