Validation of Reference Genes for Expression Studies during Craniofacial Development in Arctic Charr

Abstract

Arctic charr (Salvelinus alpinus) is a highly polymorphic species and in Lake Thingvallavatn, Iceland, four phenotypic morphs have evolved. These differences in morphology, especially in craniofacial structures are already apparent during embryonic development, indicating that genes important in the formation of the craniofacial features are expressed differentially between the morphs. In order to generate tools to examine these expression differences in Arctic charr, the aim of the present study was to identify reference genes for quantitative real-time PCR (qPCR). The specific aim was to select reference genes which are able to detect very small expression differences among different morphs. We selected twelve candidate reference genes from the literature, identified corresponding charr sequences using data derived from transcriptome sequencing (RNA-seq) and examined their expression using qPCR. Many of the candidate reference genes were found to be stably expressed, yet their quality-rank as reference genes varied considerably depending on the type of analysis used. In addition to commonly used software for reference gene validation, we used classical statistics to evaluate expression profiles avoiding a bias for reference genes with similar expression patterns (co-regulation). Based on these analyses we chose three reference genes, ACTB, UB2L3 and IF5A1 for further evaluation. Their consistency was assessed in an expression study of three known craniofacially expressed genes, sparc (or osteonectin), matrix metalloprotease 2 (mmp2) and sox9 (sex-determining region Y box 9 protein) using qPCR in embryo heads derived from four charr groups at three developmental time points. The three reference genes were found to be very suitable for studying expression differences between the morphotypes, enabling robust detection of small relative expression changes during charr development. Further, the results showed that sparc and mmp2 are differentially expressed in embryos of different Arctic charr morphotypes.

The developmental transcriptome of contrasting Arctic charr (Salvelinus alpinus) morphs

“…10,000 years, ii) of the availability of abundant AC material and iii) we wanted an extreme contrast, because of budget reasons we could only sequence 8 samples at the time. This rather extreme contrast is justified as the data and other studies 58, 59 building on this data illustrate (see discussion).…”

“…We set up crosses and reared embryos in the laboratory as previously described in 58. Embryos from four charr morphs were studied: an aquaculture charr (AC-charr) from the Holar breeding program 57 and three natural morphs from Lake Thingvallavatn; small benthivorous (SB), large benthivorous (LB) and small planktivorous (PL) charr 60 .…”

“…For each combination of morphs and timepoints we pooled RNA from approximately six individuals. RNA extraction and following steps were performed as described earlier 58, 62 . The embryos were dechorionated and homogenized with a disposable Pellet Pestle Cordless Motor tissue grinder (Kimble Kontes, Vineland, NJ, USA) and RNA was extracted into two size-fractions using the Ambion mirVana kit (Life Technologies, Carlsbad, CA, USA).…”

“…Embryos were dechorionated and decapitated in front of the pectoral fin. RNA extraction and cDNA preparation were performed as described previously in 58. Similarly, RNA was extracted from a small piece (approximately 2 mm 2 ) of skin, heart, liver, gill, spleen, intestine and liver from six adult AC-charr.…”

“…We previously identified suitable reference genes to study Arctic charr development 58 . Here we examined the expression of several genes in whole charr embryos, embryonic heads and adult tissues.…”