Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• Studying complex host-pathogen interactions requires bacterial RNA amplification when recovery is limited.
• Investigated two RNA amplification strategies for Mycobacterium tuberculosis using whole-genome microarrays.

Purpose of the Study:

• To evaluate two bacterial RNA amplification methods for their reproducibility, sensitivity, and representational bias.
• To assess the effectiveness of amplified RNA in capturing biologically relevant gene expression changes in Mycobacterium tuberculosis.

Main Methods:

• RNA amplification using oligo-dT primers after polyadenylation.
• RNA amplification using mycobacterial amplification-directed primers and T7 polymerase in vitro transcription.
• Analysis of amplified RNA from varying total RNA inputs (500, 50, and 5 ng) using whole-genome microarrays.

Main Results:

• Both amplification methods reproducibly amplified mycobacterial RNA from as little as 5 ng total RNA.
• Amplified RNA accurately reflected differential gene expression patterns observed with unamplified RNA during shifts in growth conditions (aerobic to microaerophilic).
• No significant representational bias was observed in the amplified products.

Conclusions:

• Developed two reproducible bacterial RNA amplification methods.
• These methods enable whole-genome RNA profiling for exploring host-pathogen interactions in bacterial infections.
• Facilitates previously intractable studies of bacterial gene expression during infection.