Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR

• The expression stability of housekeeping genes was evaluated in G. lamblia by RT-qPCR.
• ALD and NADH are suitable as reference genes in the WtWB strain of G. lamblia.
• GAPDH is appropriate as a reference gene in the MTZ-resistant strain of G. lamblia.
• PPDK and VSP are suitable as reference genes in the NTZ-resistant strain of G. lamblia.

The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia.