Using single-worm RNA sequencing to study C. elegans responses to pathogen infection.

Area of Science:

• Molecular Biology
• Genomics
• Developmental Biology

Background:

• Caenorhabditis elegans is a widely used model organism for biological research.
• Individual variations within isogenic populations are often overlooked, potentially masking biological impacts.
• Single-worm RNA sequencing (RNA-seq) offers a powerful approach to study these individual differences, but the technique requires further development and validation.

Purpose of the Study:

• To develop and optimize a single-worm RNA-seq method for transcriptome profiling in C. elegans.
• To assess the quality and effectiveness of the developed single-worm RNA-seq technique.
• To apply the method to investigate gene expression changes in individual worms during pathogen infection.

Main Methods:

• A five-step single-worm RNA-seq protocol was established, including mechanical lysis, RNA extraction, cDNA synthesis, library preparation, and sequence data analysis.
• Mechanical lysis using a Qiagen TissueLyser was employed to maintain RNA integrity.
• Quality control metrics were used to compare single-worm libraries with standard RNA-seq libraries.

Main Results:

• The mechanical lysis method effectively preserved RNA integrity.
• Single-worm RNA-seq libraries demonstrated comparable quality to standard RNA-seq libraries.
• Analysis of pathogen infection revealed a core set of genes and biological processes related to immune response and metabolism affected by infection at the individual level.

Conclusions:

• A robust single-worm RNA-seq method for C. elegans gene expression profiling was successfully developed.
• The method effectively captured both shared and individual-specific gene expression patterns during pathogen infection.
• Combining single-worm and standard RNA-seq approaches enables the detection of population-wide and individual-specific gene activities in isogenic populations.