Selection of suitable reference genes for gene expression studies in myxosporean (Myxozoa, Cnidaria) parasites

Abstract

Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species.

Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease

“…Owing to the presence of PCR inhibitors in bryozoan tissue, bryozoan total RNA was treated with a PowerClean Pro RNA Clean-up kit (Cambio) to remove free and nucleic acid-bound PCR inhibiting substances. PCR inhibition has been highlighted as a potential cause of low representation of myxosporean transcripts in qPCR studies, especially based on invertebrate tissue samples 74 . In our hands, we have found the PowerClean Pro RNA Clean-up kit to be highly effective in releaving PCR inhibition without the need to dilute cDNA samples (J.W.…”

Identification of reference genes for quantitative expression analysis in Indian catfish,Clarias magur, under physiological and pathological conditions

“…in numerous experiments: larval development in Senegalese sole and Atlantic halibut (Infante et al, 2008), bacterial infection in Japanese flounder (Zheng & Sun, 2011), salinity stress in spotted seabass (Wang et al, 2018), developmental stages in zebra fish (McCurley & Callard, 2008) and loach (Chen et al, 2017) and gonadal development in rice eel (Hu et al, 2014). Conversely, some studies have found GAPDH as a stable RG in different embryonic developmental stages of half-smooth tongue sole (Liu et al, 2014) and magur (Mir et al, 2018), and during myxosporean infection in rainbow trout (Kosakyan et al, 2019). In our study, GAPDH expression varied widely across the tissues.…”

Minicollagens play the governing role in polar capsule formation in parasitic cnidarians, the Myxozoa

“…Primer efficiency of each primer pair (Table S1) was evaluated by qPCR on serial cDNA dilutions and was confirmed to be above 95 % and below 110 %. Housekeeping gene selection and validation was performed as previously described (Kosakyan et al, 2019). All gene expression levels were normalised to the levels of β-actin and GAPDH housekeeping genes using the standard curve method and expressed as either fold change (log 10 ) or relative gene expression values and visualised in GraphPad (ver.…”