Validation of reference genes for expression studies during craniofacial development in arctic charr.

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

Ranking of reference gene candidates, based on stability of expression in Arctic charr whole embryos using either qPCR or RNA-seq.

<p>Abbreviations: Nf _ NormFinder, SD = standard deviation, C.V. = coefficient of variation (used to rank), p M = significant differences between morphological groups, p T = significant differences between developmental time points.</p><p>**p = <0.01; * = p<0.05; - = no significant difference.</p

The reference genes selected in this study, their abbreviation and putative function.

<p>The reference genes selected in this study, their abbreviation and putative function.</p

A scheme of sampling and analyses of Arctic charr development.

<p>(<b>A</b>) Embryos were collected at the indicated relative age (represented by vertical lines). Either whole embryos or heads of the indicated charr groups were used for RNA extraction (LB: large benthivorous charr; SB: small benthivorous charr; PL: planktivorous charr; AC: aquaculture charr). The numbers (in boxes, circles and diamonds) indicate the number of individuals pooled for each extraction. The RNA was reverse-transcribed and the cDNA used for qPCR or transcriptome sequencing as shown. Bars at top display approximate time points of cartilage formation, ossification and hatching (unpublished data). (<b>B–C</b>) Ventral and lateral views of a planktivorous head at 336 τ_s with a dashed line representing the decapitation line in front of the pectoral fin for head sample collection. Embryos were stained with alcian blue (for cartilage) and alizarin red (for bone) according to the described method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066389#pone.0066389-Javidan1" target="_blank">[74]</a> with some alterations.</p

Ranking of the candidate reference genes in Arctic charr head homogenates using BestKeeper (BK), geNorm (gN), NormFinder (Nf) and standard deviation (SD).

<p>Ranking of the candidate reference genes in Arctic charr head homogenates using BestKeeper (BK), geNorm (gN), NormFinder (Nf) and standard deviation (SD).</p

Comparison between expression values from RNA-seq and qPCR.

<p>Reads per million aligned per kilobase (RPMK) transformed to a log2-scale were plotted against equivalent Cq-values for eleven candidate reference genes. The compared samples were from the same groups and at the same or similar relative age (insert, black spots represent samples used for analysis). The line is a least squares linear fit to the data (y = 26.23–0.97x, R² = 0.815).</p

Comparison of different reference genes for normalising the expression of sox9a, mmp2 and sparc in charr heads at three embryonic stages.

<p>Expression of sox9a, mmp2 and sparc was examined with qPCR and normalised using either individual or two combinations of reference genes. Normalisation factors (NF) were based on geometric means of either two or three genes (NF = 2: ACTB and IF5A1; NF = 3: ACTB, IF5A1 and UB2L3). In each analysis (panel column) relative expression levels of the three genes in small benthivorous (SB), planktivorous (PL) and large benthivorous charr (LB), are compared to expression levels in aquaculture (AC) charr (horizontal line) at the same embryonic stage. Statistical differences of SB or LB gene expression versus expression in either PL (black circles) or AC (white circles) are indicated. Error bars represent standard deviation calculated from two biological replicates. Each biological replicate contains homogenate of six heads.</p

Expression levels of reference genes in the head of four charr groups during development.

<p>Expression profiles of 12 candidate reference genes based on quantitative real time PCR performed on embryonic heads from four charr groups at the relative ages 150 to 434 τ_s. Expression levels are shown as mean Cq (quantification cycle) values in the four charr groups at corresponding relative age, except for the two last time points (dashed line), which are based on samples of only two groups(AC and PL charr). Error bars represent standard deviation.</p