Abstract
The northern root-knot nematode (Meloidogyne hapla) is a critical pathogen with a wide host range. Quantitative real-time polymerase chain reaction (qRT-PCR) has been used to elucidate gene expression and function of M. hapla. Suitable reference genes are required to ensure accurate results of qRT-PCR for normalising gene expression. Eleven candidate reference genes of M. hapla were selected to evaluate gene expression stability under different conditions. The stability of candidate reference genes was ranked using RefFinder analysis, and the optimal number of reference genes was recommended with geNorm. Notably, the most stable reference genes were SDHA, Mdh, and RpS6 for all samples; SDHA and RpS6 were particularly stable during development stage treatments, whereas Mdh and RpS6 were appropriate for temperature and inorganic compound treatments. In contrast, the least stable reference genes were Actin1 during development stages and all other treatments, GAPDH for temperature treatments, and α-Tub for inorganic compound treatments. One target gene, Mh-Hsp90, was used to verify the selection of reference genes, results showed Mdh and RpS6 could be used as suitable reference genes for M. hapla, and Mdh plus RpS6 were better. Our finding contributes to further work on gene transcription analysis in M. hapla.
Highlights
Quantitative real-time polymerase chain reaction is an important conventional method for measuring gene expression in molecular biology applications and has several advantages, including high sensitivity, wide dynamic range, and low cost [1,2,3,4]
The E% values for the eleven candidate reference genes ranged from 93.6% to 106.7%, and the R2 values reached 0.99 (Table 1)
Α-Tub, Ubp, and EF1-α were identified as stable genes using BestKeeper analysis. These results suggested that Mdh and ribosomal protein S6 (RpS6), were the most stable genes, whereas EF1-α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were the least stable genes by RefFinder (Table 2)
Summary
Quantitative real-time polymerase chain reaction (qRT-PCR) is an important conventional method for measuring gene expression in molecular biology applications and has several advantages, including high sensitivity, wide dynamic range, and low cost [1,2,3,4]. Experimental error can be caused by poor quality and low concentrations of RNA and cDNA [5,6,7,8,9,10]. In order to reduce error and achieve reliable results, reference genes, called housekeeping genes, are essential for normalising gene expression [11]. Several common reference genes, including arginine kinase (AK), Actin 1 (Actin1), elongation factor 1 alpha (EF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), malate.
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