A methodology for global validation of microarray experiments

Miron, Mathieu; Woody, Owen Z.; Marcil, Alexandre; Murie, Carl; Sladek, Robert; Nadon, Robert

doi:10.1186/1471-2105-7-333

Cited by 66 publications

(48 citation statements)

References 28 publications

(26 reference statements)

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“…Interestingly, the SCD gene, which was selected as a control non-DE (1.5× higher expression in IB, but without statistical significance), was observed to be significantly DE in the qPCR validation step (2× upregulation in IB, P = 0.03). The substantial value of Concordance Correlation Coefficient calculated between microarray and qPCR fold-change values for these 18 genes (CCC = 0.863) indicates a remarkable agreement between both measures, validating the global reproducibility of microarray results [28]. …”

Section: Resultsmentioning

confidence: 78%

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Longissimus dorsi transcriptome analysis of purebred and crossbred Iberian pigs differing in muscle characteristics

Óvilo¹,

Benítez²,

Fernández³

et al. 2014

BMC Genomics

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BackgroundThe two main genetic types in Iberian pig production show important phenotypic differences in growth, fattening and tissue composition since early developmental stages. The objective of this work was the evaluation of muscle transcriptome profile in piglets of both genetic types, in order to identify genes, pathways and regulatory factors responsible for their phenotypic differences. Contemporary families coming from pure Iberian pigs (IB) or from crossing with Duroc boars (DU×IB) were generated. Piglets (14 from each genetic type) were slaughtered at weaning (28 days) and longissimus dorsi was sampled for composition and gene expression studies. RNA was obtained and hybridized to Affymetrix Porcine Genechip expression arrays.ResultsLoin muscle chemical composition showed significant differences between genetic types in intramuscular fat content (6.1% vs. 4.3% in IB and DUxIB animals, respectively, P = 0.009) and in saturated (P = 0.019) and monounsaturated fatty acid proportions (P = 0.044). The statistical analysis of gene expression data allowed the identification of 256 differentially expressed (DE) genes between genetic types (FDR < 0.10), 102 upregulated in IB and 154 upregulated in DU×IB. Transcript differences were validated for a subset of DE genes by qPCR. We observed alteration in biological functions related to extracellular matrix function and organization, cellular adhesion, muscle growth, lipid metabolism and proteolysis. Candidate genes with known effects on muscle growth were found among the DE genes upregulated in DU×IB. Genes related to lipid metabolism and proteolysis were found among those upregulated in IB. Regulatory factors (RF) potentially involved in the expression differences were identified by calculating the regulatory impact factors. Twenty-nine RF were found, some of them with known relationship with tissue development (MSTN, SIX4, IRX3), adipogenesis (CEBPD, PPARGC1B), or extracellular matrix processes (MAX, MXI1). Correlation among the expression of these RF and DE genes show relevant differences between genetic types.ConclusionThese results provide valuable information about genetic mechanisms determining the phenotypic differences on growth and meat quality between the genetic types studied, mainly related to the development and function of the extracellular matrix and also to some metabolic processes as proteolysis and lipid metabolism. Transcription factors and regulatory mechanisms are proposed for these altered biological functions.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-413) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 78%

“…The concordance correlation coefficient (CCC) between the fold-change values estimated in loin from microarrays and qPCR expression measures for the 18 genes was calculated to validate the global microarrays results [28]. …”

Section: Methodsmentioning

confidence: 99%

Longissimus dorsi transcriptome analysis of purebred and crossbred Iberian pigs differing in muscle characteristics

Óvilo¹,

Benítez²,

Fernández³

et al. 2014

BMC Genomics

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“…All of them were validated by qPCR (S11 Table). A concordance correlation coefficient, used to assess technical validation in high throughput transcriptomic studies [36] was calculated (CCC = 0.94) and denoted a high general concordance between RNA-Seq and qPCR expression values. In general good individual correlation values were obtained (S11 Table).…”

Section: Resultsmentioning

confidence: 99%

“…To validate the global RNA-Seq results, the concordance correlation coefficient (CCC) [36] was calculated between the FC values estimated from RNA-Seq and qPCR expression measures for the 5 genes analyzed by the two technologies (RNA-Seq and qPCR).…”

Section: Methodsmentioning

confidence: 99%

Developmental Stage, Muscle and Genetic Type Modify Muscle Transcriptome in Pigs: Effects on Gene Expression and Regulatory Factors Involved in Growth and Metabolism

Ayuso

Fernández²,

Núñez³

et al. 2016

PLoS ONE

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Iberian pig production includes purebred (IB) and Duroc-crossbred (IBxDU) pigs, which show important differences in growth, fattening and tissue composition. This experiment was conducted to investigate the effects of genetic type and muscle (Longissimus dorsi (LD) vs Biceps femoris (BF)) on gene expression and transcriptional regulation at two developmental stages. Nine IB and 10 IBxDU piglets were slaughtered at birth, and seven IB and 10 IBxDU at four months of age (growing period). Carcass traits and LD intramuscular fat (IMF) content were measured. Muscle transcriptome was analyzed on LD samples with RNA-Seq technology. Carcasses were smaller in IB than in IBxDU neonates (p < 0.001), while growing IB pigs showed greater IMF content (p < 0.05). Gene expression was affected (p < 0.01 and Fold change > 1.5) by the developmental stage (5,812 genes), muscle type (135 genes), and genetic type (261 genes at birth and 113 at growth). Newborns transcriptome reflected a highly proliferative developmental stage, while older pigs showed upregulation of catabolic and muscle functioning processes. Regarding the genetic type effect, IBxDU newborns showed enrichment of gene pathways involved in muscle growth, in agreement with the higher prenatal growth observed in these pigs. However, IB growing pigs showed enrichment of pathways involved in protein deposition and cellular growth, supporting the compensatory gain experienced by IB pigs during this period. Moreover, newborn and growing IB pigs showed more active glucose and lipid metabolism than IBxDU pigs. Moreover, LD muscle seems to have more active muscular and cell growth, while BF points towards lipid metabolism and fat deposition. Several regulators controlling transcriptome changes in both genotypes were identified across muscles and ages (SIM1, PVALB, MEFs, TCF7L2 or FOXO1), being strong candidate genes to drive expression and thus, phenotypic differences between IB and IBxDU pigs. Many of the identified regulators were known to be involved in muscle and adipose tissues development, but others not previously associated with pig muscle growth were also identified, as PVALB, KLF1 or IRF2. The present study discloses potential molecular mechanisms underlying phenotypic differences observed between IB and IBxDU pigs and highlights candidate genes implicated in these molecular mechanisms.

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“…Validation of microarray data by qRT-PCR was performed on 16 genes (10 randomly selected and 6 selected from the most differentially expressed pathways) according to the approach described by Miron et al (Miron et al, 2006). Microarray data have been deposited into the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/projects/geo/).…”

Section: Methods and Statisticsmentioning

confidence: 99%

Molecular Profiles of Parvalbumin-Immunoreactive Neurons in the Superior Temporal Cortex in Schizophrenia

Pietersen¹,

Mauney²,

Kim³

et al. 2014

Journal of Neurogenetics

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Dysregulation of pyramidal cell network function by the soma- and axon-targeting inhibitory neurons that contain the calcium-binding protein parvalbumin (PV) represents a core pathophysiological feature of schizophrenia. In order to gain insight into the molecular basis of their functional impairment, we used laser capture microdissection (LCM) to isolate PV-immunolabeled neurons from layer 3 of Brodmann’s area 42 of the superior temporal gyrus (STG) from postmortem schizophrenia and normal control brains. We then extracted ribonucleic acid (RNA) from these neurons and determined their messenger RNA (mRNA) expression profile using the Affymetrix platform of microarray technology. Seven hundred thirty-nine mRNA transcripts were found to be differentially expressed in PV neurons in subjects with schizophrenia, including genes associated with WNT (wingless-type), NOTCH, and PGE2 (prostaglandin E2) signaling, in addition to genes that regulate cell cycle and apoptosis. Of these 739 genes, only 89 (12%) were also differentially expressed in pyramidal neurons, as described in the accompanying paper, suggesting that the molecular pathophysiology of schizophrenia appears to be predominantly neuronal type specific. In addition, we identified 15 microRNAs (miRNAs) that were differentially expressed in schizophrenia; enrichment analysis of the predicted targets of these miRNAs included the signaling pathways found by microarray to be dysregulated in schizophrenia. Taken together, findings of this study provide a neurobiological framework within which hypotheses of the molecular mechanisms that underlie the dysfunction of PV neurons in schizophrenia can be generated and experimentally explored and, as such, may ultimately inform the conceptualization of rational targeted molecular intervention for this debilitating disorder.

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A methodology for global validation of microarray experiments

Cited by 66 publications

References 28 publications

Longissimus dorsi transcriptome analysis of purebred and crossbred Iberian pigs differing in muscle characteristics

Longissimus dorsi transcriptome analysis of purebred and crossbred Iberian pigs differing in muscle characteristics

Developmental Stage, Muscle and Genetic Type Modify Muscle Transcriptome in Pigs: Effects on Gene Expression and Regulatory Factors Involved in Growth and Metabolism

Molecular Profiles of Parvalbumin-Immunoreactive Neurons in the Superior Temporal Cortex in Schizophrenia

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