Determination of qPCR reference genes suitable for normalizing gene expression in a canine model of Duchenne muscular dystrophy

BACKGROUND
Dogs with dystrophin-deficient muscular dystrophy are valuable models of the equivalent human disease, Duchenne Muscular Dystrophy (DMD): unlike the mdx mouse, these animals present a disease severity and progression that closely matches that found in human patients. Canine models are however less thoroughly characterised than the established mdx mouse in many aspects, including gene expression. Analysis of expression in muscle plays a key role in the study of DMD, allowing monitoring and assessment of disease progression, evaluation of novel biomarkers and gauging of therapeutic intervention efficacy. Appropriate normalization of expression data via carefully selected reference genes is consequently essential for accurate quantitative assessment. Unlike the expression profile of healthy skeletal muscle, the dystrophic muscle environment is highly dynamic: transcriptional profiles of dystrophic muscle might alter with age, disease progression, disease severity, genetic background and between muscle groups.


OBJECTIVES
The aim of this work was to identify reference genes suitable for normalizing gene expression in healthy and dystrophic dogs under various comparative scenarios.


METHODS
Using the delta-E50 MD canine model of DMD, we assessed a panel of candidate reference genes for stability of expression across healthy and dystrophic animals, at different ages and in different muscle groups.


RESULTS
We show that the genes HPRT1, SDHA and RPL13a appear universally suitable for normalizing gene expression in healthy and dystrophic canine muscle, while other putative reference genes are exceptionally poor, and in the case of B2M, actively disease-correlated.


CONCLUSIONS
Our findings suggest consistent cross-sample normalization is possible even throughout the dynamic progression of dystrophic pathology, and furthermore highlight the importance of empirical determination of suitable reference genes for neuromuscular diseases.


Introduction
The fatal, X-linked, muscle-wasting disease Duchenne muscular dystrophy (DMD) affects roughly one in five thousand newborn boys [1], and is caused by insufficiency or absence of the muscle sarcolemma-associated structural protein dystrophin, a protein responsible for maintaining a physical link between the intracellular actin cytoskeleton and the extracellular muscle matrix environment.Loss of dystrophin weakens the sarcolemmal integrity of muscle fibres, leaving them sensitive to contraction-induced injury (especially eccentric contraction, where muscle fibres lengthen under tension) [2].As muscle tissue is highly regenerative, the disease is typically characterised by continuous cycles of muscle degeneration and regeneration: regeneration that is sufficient to retain (albeit partially-compromised) muscle performance for the first few years of life.The ongoing process of muscle damage ultimately results in a persistent inflammatory state leading to progressive loss of muscle tissue and accumulation of fatty infiltrates and fibrotic scarring.DMD boys are thus often initially asymptomatic, displaying early signs of the disease between three and five years of age, losing ambulation between the ages of eight and fourteen and losing further muscle tissue thereafter.No cure for DMD presently exists, and while improved disease management (such as a programme of anti-inflammatory corticosteroid treatment) has led to significantly increased lifespans, DMD patients today typically die in their late twenties or early thirties from either cardiac or respiratory failure [3].
Despite being an essentially monogenic condition, DMD varies considerably in severity and progression, a feature not shared by the dystrophin-deficient mdx mouse model.Dystrophin is an enormous gene with numerous splice variants: the varied site and nature of causative mutations influences the pathogenesis and also the choice of viable therapeutic approaches [4][5][6][7][8].The disease is further subject to influence from modifier genes, most of which remain poorly-characterised.Even on the comparatively consistent genetic backgrounds offered by mouse models, additional mutations in genes such as Annexin 6 substantially alter disease severity [9].In human patients, mutations reducing expression of osteopontin [10,11], or modulating signalling through TGFβ [12,13] slow disease progression, and in dogs, muscledriven expression of the Notch ligand Jagged1 is associated with retained muscle function and increased longevity [14], as is downregulation of the phosphatidylinositol protein PITPNA [15].It is likely that many thus-far undetected gene variants contribute to disease progression, and in more genetically outbred models (or indeed the enormous genetic diversity represented by the human population) such variability likely represents the norm.
Within any given individual, the precise composition of muscle tissue varies considerably as the disease progresses, and indeed the cellular milieu present within a muscle may vary over even relatively small timescales in response to atypical muscle activity.Additionally, the extent of damage, rate of disease progression, and balance of fibrotic/adipogenic replacement differs considerably between muscle groups and with frequency of muscle use.
In DMD patients, loss of ambulation invariably occurs before loss of upper limb mobility, and the diaphragm and intercostal muscles (muscle groups subject to essentially constant use and eccentric contraction) are among the most severely affected: these muscles show pronounced dystrophic pathology even in the otherwise relatively mildly-affected mdx mouse.In stark contrast, the extraocular muscles appear to be highly resistant to dystrophic pathology, retaining essentially unaltered function throughout the course of disease [16].
Taken together, this highlights the importance of ensuring that potential therapies are tested in more severely-affected, more genetically-diverse animal models such as the dog [17][18][19], but also illustrates the challenges to performing direct individual-to-individual comparison of disease progression (and response to therapeutic intervention) in such models.

Quantitative analysis of gene expression in dystrophic muscle:
Measurement of gene expression in dystrophic muscle readily allows monitoring of disease progression and response to intervention, however such assessment is hampered by the innate variability described above.While semi-quantitative RT-PCR (and even nested RT-PCR) via gel densitometry is surprisingly commonplace in the DMD field, truly quantitative gene expression data is chiefly obtained via qPCR.The accuracy of this technique is criticallydependent on good normalization: even with identical starting material, slight differences in the efficiency of RNA isolation or cDNA synthesis can significantly affect subsequent quantitation.Effective normalization requires appropriate reference genes, and indeed efforts to identify, validate and publicize such genes are becoming more common in a variety of disease states [20][21][22][23][24] and model organisms [25][26][27][28][29][30].A review of the literature specifically within the DMD field however reveals a considerable number of candidates: selected examples in dystrophic dogs include GAPDH [31][32][33], RPS18 [34], HPRT1 [35,36], 18S [37]; in humans, TBP and GUSB [13]; and in mice GAPDH [33,38], ActB [39], 18S [40].There appears to be minimal effort to apply reference genes consistently between studies (even varying from manuscript to manuscript within a research group), and data supporting the selection of the gene or genes used is rarely presented.Moreover, use of a single reference gene is common, despite MIQE guidelines [41].Use of two or three such genes is, while demonstrably more expensive and time-consuming, nevertheless necessary for the generation of highquality data (especially in more genetically outbred models such as the dog) and is thus highly recommended [42].Selection of such genes is by no means trivial even in reasonably transcriptionally-static tissues, and becomes particularly challenging under conditions where a high degree of transcriptional plasticity might be expected (such as a progressive disease state).mRNA is typically subject to a higher rate of turnover than many proteins, and as a consequence even genes regarded as canonically 'stable' at the protein level (such as GAPDH or beta-actin) can vary significantly between samples, and indeed alter in expression over even relatively short timeframes [24,43,44].This inherently dynamic behaviour of mRNA, combined with the progressive pathological changes associated with DMD, the differing severity dependent on muscle type and use, and the presence of modifier genes that worsen or ameliorate disease progression all potentially place severe restrictions on the studies that may be performed; such limitations make accurate comparisons of gene expression between individuals, between muscles, or over extended time periods, particularly difficult [45].
Determination of a set of reference genes suitable for normalizing gene expression between individuals, between muscle groups and between age groups would therefore be beneficial; a unified set of genes suitable for all these categories combined would (if possible) be especially useful.The delta-E50 MD dog model of DMD [46] offers a perfect test case for identification of such reference genes.

Determination of reference genes:
Identification of suitable reference genes for qPCR normalization is by necessity somewhat convoluted: effective validation of a candidate reference gene would classically require comparison with an existing validated reference gene, essentially presenting an infinite regression problem.Several different methodologies have been proposed to circumvent these difficulties, each assessing suitability of candidate genes by subtly different criteria, and each thus exhibiting unique strengths and weaknesses.We typically employ three of these methods, namely geNorm [47], Normfinder [48] and Bestkeeper [49]: all three require a dataset comprising gene expression data for a representative number of samples, for all the candidate reference genes, and all three use Microsoft Excel (as an executable macro, an addin and a write-protected spreadsheet, respectively) thus all three are readily accessible.
Moreover, each program determines suitable reference genes via different algorithms: genes identified as high-scoring by all three methodologies are therefore likely to be strong candidates.
A detailed consideration of the strengths and weaknesses of the three packages is provided (supplementary information 1), however the essential details and merits (and relevant links to software) are summarised below.geNorm analysis uses an iterative pairwise approach, identifying the pair of genes with the greatest pairwise correlation.The method does not assess overall expression stability, thus is tolerant of noisy datasets (but relatively sensitive to outliers).As a pairwise approach, the software identifies a minimum of two suitable genes (single candidates with greater stability will be ignored).A Microsoft-office compatible copy of the original excel macro is available at http://ulozto.net/xsFueHSA/genorm-v3-zip.
Bestkeeper analysis compares individual expression profiles for each reference gene to a geometric mean profile generated from all gene candidates; in essence ranking the extent to which individual genes reflect the behaviour of the dataset as a whole.and elements of marked diversity (inclusion of multiple muscle groups, including mandibular and extraocular muscles that in dogs uniquely express the 2M and EO myosin heavy chains, respectively).We have combined this collection of samples with a relatively large panel of reference genes (ActB, UBC, 18S, SDHA, RPL13a, YWHAZ2, B2M and HPRT1 -see table 1) to generate a dataset for use in the three software algorithms described above.Analysing this dataset in several configurations allowed us to determine appropriate reference genes for normalizing expression under various specific constraints, revealing reference genes that scored highly under all conditions investigated.

Animal cohorts and tissue collection:
Maintenance of the dystrophic delta-E50 MD dog colony and biopsy for the natural history study were conducted under Project Licence approval from the UK Home Office and following approval from the Royal Veterinary College Ethics and Welfare committee.
Nine male dogs (5 dystrophic, 4 healthy) from the colony were used for study of age-related gene expression, with biopsy samples collected from the left vastus lateralis muscle under general anaesthesia at three month intervals, up to a maximum age of eighteen months as part of a larger natural history disease characterisation.Where possible, dystrophic and healthy littermates were used for each time-point, with samples being collected as dogs of the appropriate age became available.Final coverage thus consists of: 3 months, N=8 (5:3 DMD:WT); 6 months, N=7 (3:4); 9 months, N=5 (2:3); 12 months, N=4 (2:2); 15 months, N=4 (1:3); 18 months, N=5 (2:3).One healthy dog from this cohort (G2) was subsequently euthanized at 18 months of age to provide a panel of muscle types, and D1 and D2, two additional dogs (dystrophic littermates) were added to provide a similar panel of dystrophic muscles (samples collected post-mortem following humane end-point euthanasia).As the ages of these latter dogs at euthanasia (14 and 17 months, respectively) were close to the 15 and 18 month age brackets defined above, these samples were also included in several comparisons of age-related gene expression (see supplementary figure 1 for IDs, litter matching, genotypes, muscle groups and ages).Immediately following collection, muscle samples (approximately 1cm 3 ) were mounted on cork blocks on a bed of OCT (Tissue-Tek) and snap frozen under liquid-nitrogen-cooled isopentane to preserve tissue morphology.
Frozen tissues were used for histological serial cryosectioning (as part of a separate study), with interleaving (unmounted) sections collected for isolation of the RNA used in this study (50-100 8um sections per sample, ca.40-100mg tissue).

RNA isolation and cDNA synthesis:
Frozen tissue sections were rapidly mixed with RNA-Bee reagent (Amsbio) and RNA extracted following the manufacturer's instructions (with inclusion of an additional 1:1 chloroform extraction following phase separation, and inclusion of glycogen at 10ug.ml -1 during precipitation to maximise RNA yield).RNA purity was confirmed by nanodrop (ND1000), with samples exhibiting significant guanidium carryover (260/230<1.7)further cleaned by a second isopropanol precipitation step.
cDNA was prepared from RNA using the RTnanoscript2 kit (PrimerDesign) using random 9mer and oligodT priming, with  Dystrophic muscle panel (all muscles, two ages, dystrophic only)

Normfinder:
As described above, the Normfinder algorithm can be used with datasets as a whole, but also offers the option of grouping data in a user-dependent fashion: allowing measurement of between-group and within-group variation for a potentially large number of grouping criteria.
Our Normfinder analysis therefore used two approaches: ungrouped, assessing expression variation overall, using the entire (RQ) dataset or selected subsets exactly as described for geNorm and Bestkeeper (above), or grouped, looking at variation in expression over the entire dataset (or subsets as above), between specific, user-specified groups.Note: several samples were necessarily omitted under specific grouping criteria where groups of one sample would result (for example 'by individual') as groups must contain at least two samples.This latter analysis can be particularly useful not only for identifying universally-suitable reference genes but also for isolating group-specific genes (such as a gene showing strong variation with age, or muscle type).Dataset elements and groupings were as follows:

Cq determinations:
Individual Cq values fell within a relatively consistent range for any given gene, with the greatest sample-to-sample variation observed in ActB and the least in 18S (see supplementary figure 2).With the exception of the abundantly-expressed 18S ribosomal RNA, the candidate genes examined covered a total Cq range of over 13 cycles, equivalent to differences in mRNA expression of around 4 orders of magnitude.

GeNorm analysis:
The iterative pairwise approach of the geNorm algorithm ranks genes by average expression stability (M), where lower scores represent higher stability.As shown (table 2, figure 1 and supplementary figure 3), RPL13a, HPRT1, 18S and SDHA were universally scored as the highest ranking genes (lowest expression stability value), and indeed RPL13a and HPRT1 were ranked as the highest scoring pair by the geNorm algorithm for all but one of the dataset combinations (the only exception provided by the dataset of healthy quadriceps samples alone, where RPL13a was paired with 18S instead, with HRPT1 falling to the next highest rank).The commonly accepted geNorm threshold for suitable reference gene stability is M<0.5 (bold gene names in table 2, dashed line in figure 1 and supplementary figure 3), thus while 18S and SDHA fall just outside this threshold under several dataset combinations, RPL13a and HPRT1 are consistently ranked as suitable reference genes by the geNorm algorithm.Interestingly, by this metric, fully 6 of the 8 genes were ranked as suitable for our healthy muscle panel (figure 1b), though ActB (beta-actin) scored comparatively poorly under all dataset combinations, even those assessing healthy muscle alone.
The geNorm algorithm also calculates the pairwise variation arising from inclusion of additional reference genes, in essence indicating whether the 'best pair' is sufficient, or whether better normalization can be obtained by use of 3 or more genes.In all cases, increasing number of reference genes to 3 or 4, lowered overall variation (supplementary figure 4), however values below 0.2 are considered acceptable [47], and the suggested pair of genes was sufficient to pass this threshold in every instance.

Bestkeeper analysis:
The Bestkeeper spreadsheet generates a large quantity of data, however the most useful output metric tends to be the coefficient of correlation (r): the extent of correlation between a given reference gene and the 'bestkeeper' (a composite derived from all candidate reference genes), where higher values represent greater correlation.In essence, this value reveals the individual gene that best reflects the behaviour of the dataset as a whole.As shown (table 3, figure 2, supplementary figure 5), HPRT1 and SDHA tended to rank highly under most combinations, with RPL13a also scoring highly (though usually of lower rank).In marked contrast to geNorm analysis, YWHAZ2 and B2M also tended to perform well.There is no established threshold Bestkeeper correlation value for 'good' reference genes (thresholds shown in table 3 are for comparative purposes only), however the data shows a tendency toward a sharp divide between low and high correlation (figure 2, supplementary figure 5): poor candidates are very poor, while often 5 or more of the remaining genes score comparatively highly.
As with geNorm, ActB was near-universally scored as the least suitable reference gene.Only UBC and 18S displayed comparably poor suitability.Also in agreement with geNorm analysis, when our healthy muscle panel was analysed alone (figure 2b), the majority of our candidate genes (all except ActB and 18S) showed high correlation with the 'bestkeeper' (r > 0.85).

Normfinder analysis:
Ungrouped analysis (table 4, figure 3 and supplementary figure 6), assessing overall expression stability in the entire dataset, or in subsets as for geNorm and Bestkeeper (above) revealed that SDHA and HRPT consistently scored highly (low stability value) under essentially all combinations examined, with YWHAZ2 also performing well (especially under dystrophic conditions alone).As with the geNorm and Bestkeeper analyses, ActB was again nearuniversally ranked as the least appropriate candidate, however (similar to Bestkeeper, but in marked contrast to its ranking under geNorm), RPL13a here often ranked comparatively poorly (with the exception of the healthy muscle panel).As with Bestkeeper analysis, there is no conventional threshold value for Normfinder suitability, however regardless of absolute value, a stark difference was observed between ActB and essentially every other candidate gene: the 'poor score' for RPL13a was thus typically only marginally worse than the 'high score' for SDHA.An examination of absolute stability values also reiterates the transcriptional homogeneity of our healthy muscle panel suggested by geNorm and BestKeeper analysis: fully 5 of our 8 candidate genes exhibited values below 0.2, something no single gene was able to achieve in the matching dystrophic muscle panel (figure 3b).
Under grouped analysis (table 5, supplementary figure 7, supplementary table 1), a more mathematically-sophisticated comparison assessing both intergroup and intragroup variation, a similar pattern was observed.Again SDHA and HPRT1 scored highly essentially regardless of grouping criteria, while RPL13a was less favoured.Normfinder grouped analysis additionally provides a 'best pair': a pair of genes that in combination outperform the highest scoring single candidate (note: these genes need not themselves be the highest scoring individually -a pair of genes that vary considerably, but in opposite directions, can combine to provide more accurate normalization).In almost every instance, SDHA or HPRT1 comprised one member of the 'best pair', further supporting the suitability of these reference genes.We note however that these genes were rarely suggested together: instead YWHAZ2 was near universally suggested as a partner.This gene also notably scored reasonably highly under all combinations and groupings save those of healthy dog samples alone.

Further validation:
HPRT1 and SDHA ranked highly under essentially all algorithms and conditions, both in pairwise variation and stability, while RPL13a showed marked pairwise correlation with HPRT1 and a tendency to score (if not necessarily rank) highly under most conditions, especially those assessing healthy muscle alone.Conversely, Bestkeeper analyses showed a marked tendency to score B2M (beta-2-microglobulin) highly while both geNorm and Normfinder algorithms suggested the reverse, especially when comparing both healthy and dystrophic samples (tables 2, 3 and 4): notably, while showing a high correlation with the 'bestkeeper', B2M showed much lower correlation with other individual gene candidates.As geNorm assesses by pairwise comparison, and Normfinder by overall stability, the implication is that B2M exhibits low expression stability but also potent influence over the dataset as a whole: a hallmark of a strongly disease-associated gene.To confirm this hypothesis and validate our selected reference genes, B2M expression data for our natural history samples were normalized using the geometric mean of SDHA, RPL13a and HPRT1 (in essence, using high-scoring reference genes to normalize potentially disease-associated genes).As shown in figure 4a, B2M expression is significantly enhanced in dystrophic muscle, by approximately two-fold when compared with healthy muscle.
To further confirm the suitability of our nominated reference genes, we additionally measured (and normalized) expression of the myogenic transcription factors Myf5 and MEF2C -markers of actively regenerating muscle.As shown in figure 4b and c, both these genes were also strongly upregulated in dystrophic muscle, and to a similar extent as B2M.

Discussion
Canine models of muscular dystrophy are increasingly coming to represent a key translational element in the study of this disease, exhibiting disease severity and progression (and concomitant loss of muscle performance) in a manner that closely-mimics the human condition [17][18][19].As measurement of gene expression is a major component of the investigative toolset, a panel of reference genes suitable for normalizing measured expression data in this animal model under a multitude of different conditions would be highly beneficial, and would moreover support the hypothesis that a similarly broadly-applicable set of genes might be found for human samples.The data presented here strongly suggest that HPRT1 and SDHA (and to a lesser extent, RPL13a) are suitable candidate reference genes for normalizing gene expression in both healthy and dystrophic canine muscle, being consistently ranked highly when comparing muscle samples taken at different ages (and thus in dystrophic muscle, stages of disease progression), between different individuals, and even between different muscles (including selectively-spared muscle groups such as the extraocular muscles).It should be noted that 'highest ranking' need not necessarily imply 'high scoring': any panel of genes, even one comprised entirely of those known to be highly dysregulated in DMD, would be nevertheless ranked in order of expression stability by these software programs (effectively producing a ranking of 'terrible stability' to 'poor stability').Our data here shows however that not only are SDHA, HPRT1 and RPL13a near-consistently highranking, they are also consistently high-scoring: being both the favoured genes from our candidate panel, and also empirically highly stable.
This finding in itself is somewhat surprising: the transcriptional environments represented by these different samples are likely to be highly diverse, and it was by no means guaranteed that any candidate genes would be suitable under all circumstances.Indeed, even if such a set of genes existed, it was a distinct possibility that these genes would, rather than being strong reference genes, instead simply be the 'least poor' at normalizing expression within these diverse environments (with distinct condition-specific combinations of reference genes being genuine strong candidates).As our data show however, this is not the case: the nominated reference genes are of comparable stability when analysed as specific categories (age, individual, disease) as they are when employed as an entire dataset.
Interestingly, analysis of a panel of healthy muscles alone (multiple muscles from one healthy individual) yields markedly greater stability values for all candidate genes than any dystrophic or combined dataset, and greater values than a single healthy muscle type (vastus lateralis) over time.This finding highlights the stark differences in transcriptional activity between healthy and dystrophic muscle and further suggests that age plays a greater factor in transcriptional profile than muscle type: adult (healthy) muscle tissue appears transcriptionally highly conservative regardless of which muscle is examined.Even within such a stable environment, however, SDHA, HRPT1 and RPL13a remained very high-scoring candidates.
RPL13a codes for a protein component of the 60S ribosomal subunit, and thus might reasonably be expected to show high stability under resting conditions: protein synthesis being one of the few constant requirements of virtually all cells.Given the stark changes in cellular composition between healthy and dystrophic muscle, however, it is perhaps surprising that the translational demands of the tissue as a whole appear to remain consistent.One might also expect RPL13a to closely-mirror the expression of 18S ribosomal RNA (a common choice of reference gene [37,40]): while the two are indeed scored closely under several conditions, this is not universal.Moreover, only the protein and the rRNA need strictly correlate: as an mRNA, RPL13a is subject to further amplification at the translation stage, while 18S ribosomal RNA is not.As our study shows (see Cq values, supplementary figure 2), while expression of RPL13a is the highest measured for all candidate mRNAs, it is still lower in absolute expression than 18S by almost 2 orders of magnitude.
HPRT1 codes for hypoxanthine phosphoribosyltransferase 1, an enzyme involved in purine biosynthesis via the salvage pathway [51].While such an important function readily suggests a housekeeping role, the implication that metabolic requirements for purines are similar between healthy and dystrophic muscle is an unexpected finding (the former being largely a post-mitotic tissue, the latter a highly dynamic regenerative environment).The authors note that this gene has been proposed as a suitable reference gene by others [35,36].
SDHA (succinate dehydrogenase subunit A) represents a more classical finding, being a ubiquitous mitochondrial enzyme component and thus also a gene strongly associated with mitochondria-rich skeletal muscle.Expression of this gene appears very stable, particularly over time: all our quadriceps biopsy samples exhibit near-uniform expression regardless of disease, individual or age.This was unexpected: dystrophic muscle is subject to pathological wasting as disease progresses, thus the percentage of a given dystrophic sample that is functional muscle necessarily falls with age.It is however widely recognised that the more highly-oxidative fibres (i.e.type I) are selectively spared as disease progresses, with losses primarily incurred by the faster glycolytic fibres (2A and in particular, 2X) [52].Our data here thus suggests not only that SDHA is a suitable reference gene, but also by implication that regardless of fibre loss, the oxidative capacity of dystrophic muscle (as measured by SDHA expression) does not significantly decrease over time in this model.
The samples used to prepare our dataset were selected to offer relatively comprehensive coverage of potential variability, and thus included healthy and diseased tissue, tissues of different ages, taken from different individuals, and including multiple muscle groups.A number of caveats must nevertheless be acknowledged: the multiple muscle groups included, while extensive, were derived from only 3 individual animals (D1, D2 and G2), two of whom were dystrophic littermates.These samples comprise more than half our entire sample set, thus our 'total' dataset is inherently weighted toward these 3 individuals, and toward dystrophic muscle as a whole (though this latter is perhaps less contentious given the focus of this work).Similarly, our panel of age-matched healthy/dystrophic biopsy samples, assessing disease progression over time, are exclusively derived from a single muscle (the vastus lateralis) thus our analyses ostensibly identify reference genes suitable for normalizing expression over time in this muscle alone.Lastly, due to the relatively nascent nature of our delta-E50 MD dog colony, only 5 animals (3 healthy, 2 dystrophic) were of sufficient age to contribute to the full course of age-selected muscle samples: younger samples are thus over-represented (see supplementary figure 1).These limitations accepted, we nevertheless feel that our panel of samples provides a good coverage of the most likely sources of variation when analysing dystrophic/healthy muscle, and that our conclusions regarding the broad utility of our reference gene candidates are thus justified.
A puzzling finding was the tendency of Normfinder analysis to rank RPL13a among the lowest scoring genes, while concomitantly nevertheless agreeing with Bestkeeper and geNorm as to the suitability of SDHA and HPRT1.Indeed, via the pairwise ranking of geNorm, RPL13a and HPRT1 were almost universally placed as the most closely-correlated pair.As shown in figure 5, the 2 genes exhibit highly-correlated expression values for the dataset (Spearman's rho correlation of 0.87, compared to 0.57 and 0.47 for HPRT1/SDHA and RPL13a/SDHA respectively), thus one might expect these 2 to be ranked similarly by all 3 software algorithms.As noted above however, our Normfinder analysis tended simply to single out ActB: this gene was near-consistently placed last, and by a considerable margin, with the other candidate genes tending to be relatively close-matched in stability (see figure 3 and supplementary figures 5 and 6).The Normfinder algorithm scores genes by individual stability of expression (rather than by pairwise comparison to other genes, or to a gestalt of all genes examined) thus unlike geNorm or Bestkeeper, stability and correlation are not intrinsically linked.Minor variations in overall expression stability could well account for the lower score of RPL13a despite its clear pairwise correlation with HPRT1, and we do not thus consider the poor Normfinder performance of this gene to be sufficient grounds for discarding it from our lead candidates.All 3 algorithms thus appear (with caveats) to reach near-uniform consensus regarding strong reference gene candidates, and furthermore all 3 methods also scored ActB (beta-actin) as the least suitable candidate under essentially all conditions.This gene exhibited the widest range of Cq values (supplementary figure 2), and we have previously shown ActB to be an exceptionally poor reference gene in cultured differentiating myoblasts derived from healthy and dystrophic mice [27].Our data here thus supports the hypothesis that ActB is a poor choice of reference gene for muscle-derived samples as a whole (though we also note this gene has been employed as a reference in mdx mice [39]).
Beyond simple identification of suitable reference genes (and concomitantly, poor reference genes), an advantage of using such a broad sample set is the potential to identify conditionspecific gene regulation.While the pairwise correlation algorithm of geNorm is less useful in this respect (as genes are never considered in isolation), the individual gene stability assessment of Normfinder (and the facility for user-determined grouping) is potentially very powerful.Our analysis allowed us to single out B2M (beta-2-microglobulin) as a candidate gene likely to exhibit disease-specific changes in expression, and further (by normalizing with our high scoring reference genes) allowed us to confirm that this gene is upregulated by approximately 2-fold in dystrophic muscle, thus illustrating the power of our approach.While such a DMD-specific upregulation has not to our knowledge been previously reported (though B2M has been noted to be a poor scoring reference gene in the muscle of mdx mice, along with ActB [38]), this finding is perhaps not entirely surprising: B2M is (in humans and mice) canonically relatively poorly-expressed in muscle, but highly expressed in immune lineages [53,54], thus the persistent inflammatory state characteristic of dystrophic muscle may well lead to a marked enhancement of B2M gene expression.
To further confirm the validity of our selected reference genes, we measured expression of the basic helix-loop-helix (bHLH) myogenic factor Myf5 and the myocyte enhancer factor MEF2C in our natural history muscle samples.While both of these genes are expressed in healthy muscle (MEF2C is constitutively expressed by myonuclei, and Myf5 is found in the satellite cell population and in muscle spindles [55,56]), these transcription factors are strongly induced during myogenesis and would thus be expected to be significantly upregulated in muscle undergoing active regeneration.As expected, both these genes (after normalization) exhibit highly significant dystrophy-associated increases in expression, to an extent comparable with B2M.
We thus present the encouraging finding that, for the purposes of normalizing gene expression in dystrophic and healthy dog muscle -regardless of muscle type, individual or age-the genes SDHA, HPRT1 and RPL13a appear universally suitable, and that several other candidate genes are not only unsuitable, but even actively disease-associated.Our work thus also illustrates the importance of empirically determining suitable reference genes for quantitative analysis, and addresses the limitations and advantages of the various software algorithms a researcher might employ toward this end.
While this discovery should prove of considerable benefit to further studies of this animal model in particular, we cannot at present confirm whether these findings extend to other canine models such as the GRMD dog.The fact that these genes are high scoring under such varied conditions would tend to support this assertion, however: we deem it unlikely that expression differences between two breeds of Canis familiaris would surpass that between young healthy muscle and aged dystrophic muscle.
A wider revelation of this work is that such broadly-applicable reference gene candidates exist at all (and can be empirically determined).While we stress that our data should not be taken to imply that HPRT1, RPL13a and SDHA are appropriate for human samples, our data nevertheless support the hypothesis that a similar trio of genes might be found for            Least stabl e

Figure 1 :
Figure 1: geNorm analysis Representative outputs of the geNorm algorithm.geNorm ranking (left to right: least stable to most stable) for the entire dataset (a); or for our healthy muscle panel only (b).Dashed line indicates accepted threshold for use as a reference gene (M<0.5).

Figure 2 :
Figure 2: BestKeeper analysis Representative outputs of the BestKeeper algorithm.Coefficient of correlation values for the reference gene candidates are shown for the entire dataset (a) or healthy muscle panel (b) samples, ranked (left to right) from least stable to most stable.

Figure 3 :
Figure 3: Normfinder analysis (ungrouped) Representative outputs of the Normfinder algorithm.Stability values (left to right: least stable to most stable) for the reference gene candidates are shown for the entire dataset (a) or for our healthy muscle panel (b).

Figure 5 :
Figure 5: Correlation in relative gene expression of high scoring candidates Correlation between HPRT1, SDHA and RPL13a for all muscle samples in our dataset (Spearman's rho).HPRT1 and RPL13a are very strongly correlated.

Figure
Figure 2

Figure 5 favours
Figure 5 Analysis was performed on the dataset as a whole, or using subsets of data as described below.
qPCR reactions were performed in duplicate or triplicate with 2ul cDNA per well (approx 8ng), using PrecisionPLUS SYBR green mastermix (PrimerDesign) with primers to ActB, UBC, 18S, SDHA, RPL13a, YWHAZ2, B2M and HPRT1 (see table1) taken from the geNorm Canis familiaris set (PrimerDesign), and primers to Myf5 and MEF2C designed using primer3 software (http://primer3.ut.ee/).PCR was conducted in a CFX384 light cycler (BioRad) with Cq values determined via regression.the Windows 7 operating system and Excel 2003 or 2010 (Microsoft).Bestkeeper analysis used raw Cq values, while for geNorm and NormFinder, values for each gene were linearized by conversion to relative quantities (RQ). Natural history dystrophic samples (dystrophic vastus lateralis, all ages)  Natural history healthy samples (non-dystrophic vastus lateralis, all ages)  Dystrophic samples (all muscles, all ages, dystrophic only)  Healthy samples (all muscles, all ages, healthy only)  Muscle panel (all muscles, two ages)  Healthy muscle panel (all muscles, single age, healthy only)

Table 5 :
Normfinder rankings (grouped) Normfinder results for the entire dataset or healthy/diseased subsets grouped by different criteria (as indicated: top row; datasets, second row; criterion), ranked from highest scoring (lowest stability value) to lowest scoring.Grouped analysis also suggests the best pair of genes for normalization (third row), (not necessarily the highest scoring individually).Bold: stability <0.4; italics: stability > 0.8 dk/normfinder-software