Quantitative evaluation of endogenous reference genes for ddPCR under salt stress using a moderate halophile

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Droplet digital PCR (ddPCR) offers superior sensitivity and specificity for gene detection.
• Accurate gene expression analysis under salt stress necessitates the use of endogenous reference genes (RGs).

Purpose of the Study:

• To select and validate suitable endogenous reference genes (RGs) for gene expression studies in halophiles under salt stress using ddPCR.
• To identify the most stable RG for normalizing gene expression data in Alkalicoccus halolimnae exposed to varying salt concentrations.

Main Methods:

• Six candidate RGs were selected from quantitative proteomics data of A. halolimnae across four salinity levels.
• RG expression stability was assessed using geNorm, NormFinder, BestKeeper, and RefFinder algorithms.
• The pdp gene was identified as the most stable RG and used to normalize target genes (ectA, ectB, ectC, ectD).

Main Results:

• The pdp gene exhibited minimal fluctuation in cycle threshold (Ct) values and copy numbers across different salinities.
• pdp demonstrated superior expression stability compared to other candidate RGs across all evaluated algorithms.
• Normalization using the single RG pdp provided reliable gene expression quantification under salt stress.

Conclusions:

• The pdp gene is the most suitable endogenous reference gene for quantitative gene expression analysis in A. halolimnae under salt stress conditions.
• This study provides a foundational framework and methodological reference for identifying internal controls in ddPCR-based stress response studies for halophiles.
• The findings are crucial for advancing the understanding of salt stress mechanisms in extremophilic bacteria.