Microgenomic Analysis in Skeletal Muscle: Expression Signatures of Individual Fast and Slow Myofibers
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{"title"=>"Microgenomic analysis in skeletal muscle: Expression signatures of individual fast and slow myofibers", "type"=>"journal", "authors"=>[{"first_name"=>"Francesco", "last_name"=>"Chemello", "scopus_author_id"=>"40560898000"}, {"first_name"=>"Camilla", "last_name"=>"Bean", "scopus_author_id"=>"7006260972"}, {"first_name"=>"Pasqua", "last_name"=>"Cancellara", "scopus_author_id"=>"25824648400"}, {"first_name"=>"Paolo", "last_name"=>"Laveder", "scopus_author_id"=>"6506687662"}, {"first_name"=>"Carlo", "last_name"=>"Reggiani", "scopus_author_id"=>"7006474608"}, {"first_name"=>"Gerolamo", "last_name"=>"Lanfranchi", "scopus_author_id"=>"26642916600"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-79952055409", "sgr"=>"79952055409", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0016807", "pmid"=>"21364935", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pui"=>"361337222"}, "id"=>"ff59b8e9-c5af-3816-bd26-c79093c51855", "abstract"=>"BACKGROUND: Skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: We have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification.\\n\\nCONCLUSIONS/SIGNIFICANCE: As gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions.", "link"=>"http://www.mendeley.com/research/microgenomic-analysis-skeletal-muscle-expression-signatures-individual-fast-slow-myofibers", "reader_count"=>76, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>4, "Researcher"=>13, "Student > Doctoral Student"=>9, "Student > Ph. D. Student"=>25, "Student > Postgraduate"=>5, "Student > Master"=>11, "Other"=>1, "Student > Bachelor"=>4, "Professor"=>4}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>4, "Researcher"=>13, "Student > Doctoral Student"=>9, "Student > Ph. D. Student"=>25, "Student > Postgraduate"=>5, "Student > Master"=>11, "Other"=>1, "Student > Bachelor"=>4, "Professor"=>4}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>12, "Nursing and Health Professions"=>1, "Agricultural and Biological Sciences"=>50, "Medicine and Dentistry"=>6, "Neuroscience"=>1, "Sports and Recreations"=>2, "Social Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>6}, "Neuroscience"=>{"Neuroscience"=>1}, "Social Sciences"=>{"Social Sciences"=>1}, "Sports and Recreations"=>{"Sports and Recreations"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>50}, "Nursing and Health Professions"=>{"Nursing and Health Professions"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>12}, "Unspecified"=>{"Unspecified"=>2}}, "reader_count_by_country"=>{"Colombia"=>1, "United States"=>2, "Ireland"=>1, "Brazil"=>1, "United Kingdom"=>1, "Portugal"=>1, "Germany"=>1, "Spain"=>1}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/797740"], "description"=>"<p>Pathway analysis of 4,555 significant genes identified by two-class SAM analysis (FDR about 5%). Each pathway is associated to number of genes (count) and P-values (the lower, the better). Additional information in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016807#pone.0016807.s006\" target=\"_blank\">Dataset S4</a>.</p>", "links"=>[], "tags"=>["signaling", "pathways", "kegg", "bioinformatics"], "article_id"=>468109, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016807.t003", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Metabolic_and_signaling_pathways_identified_at_the_KEGG_bioinformatics_resource_/468109", "title"=>"Metabolic and signaling pathways identified at the KEGG bioinformatics resource.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-02-22 02:15:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/797665"], "description"=>"<p>Functional classification of 5,018 DE genes identified by one-class SAM analysis (FDR below 0.25%). GO enrichment was performed with the GOTM tool: general categories were identified, which are shown in bold letters and are associated to P-values (the lower, the better). Several sub-categories were further identified with the DAVID tool, which are associated to a score number (the higher, the better). Additional information in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016807#pone.0016807.s004\" target=\"_blank\">Dataset S2</a>.</p>", "links"=>[], "tags"=>["classification", "de", "genes", "one-class", "sam"], "article_id"=>468030, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016807.t001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Functional_classification_of_DE_genes_identified_by_one_class_SAM_analysis_/468030", "title"=>"Functional classification of DE genes identified by one-class SAM analysis.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-02-22 02:13:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/398449", "https://ndownloader.figshare.com/files/398461", "https://ndownloader.figshare.com/files/398469", "https://ndownloader.figshare.com/files/398478", "https://ndownloader.figshare.com/files/398499", "https://ndownloader.figshare.com/files/398516"], "description"=>"<div><h3>Background</h3><p>Skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ.</p> <h3>Methodology/Principal Findings</h3><p>We have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification.</p> <h3>Conclusions/Significance</h3><p>As gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions.</p> </div>", "links"=>[], "tags"=>["microgenomic", "skeletal", "signatures", "myofibers"], "article_id"=>138709, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0016807.s001", "https://dx.doi.org/10.1371/journal.pone.0016807.s002", "https://dx.doi.org/10.1371/journal.pone.0016807.s003", "https://dx.doi.org/10.1371/journal.pone.0016807.s004", "https://dx.doi.org/10.1371/journal.pone.0016807.s005", "https://dx.doi.org/10.1371/journal.pone.0016807.s006"], "stats"=>{"downloads"=>23, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Microgenomic_Analysis_in_Skeletal_Muscle_Expression_Signatures_of_Individual_Fast_and_Slow_Myofibers/138709", "title"=>"Microgenomic Analysis in Skeletal Muscle: Expression Signatures of Individual Fast and Slow Myofibers", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-02-22 02:25:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/797039"], "description"=>"<p><b>A</b>) Dendogram obtained by hierarchal clustering of expression data generated by 10 pure fibers expressing MyHC-1 (soleus) and 10 pure fibers expressing MyHC-2b (EDL). Microarrays mRNA expression profiling permitted a clear distinction between type 1 and type 2B fibers. Furthermore, technical replicas grouped together within each experiment, confirming the good quality of microarray data. Analysis performed with MeV tool on the set of 11,964 probes that passed the normalization and filtering steps, using Pearson correlation distance. EDL samples came from mice number 1 (1–2), 2 (3–5), 3 (6–8) and 4 (9–10); soleus samples from mice 5 (1–2), 6 (3–5), 7 (6–7) and 8 (8–10). The letters a, b refer to spot replicates present in each microarray slide. <b>B</b>) Venn diagram formed by DE genes identified after SAM analyses. Ovals: one-class test; circles, two-class test. Overlapping areas represent genes positive to both tests. FDR values were 0.15% in the one-class test and 0.21% in the two-class test.</p>", "links"=>[], "tags"=>["microarray"], "article_id"=>467409, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016807.g002", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Statistical_analysis_of_microarray_data_/467409", "title"=>"Statistical analysis of microarray data.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-22 02:03:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/797573"], "description"=>"<p>Expression levels among individual type 2B fibers of three selected genes (JunB, Fos, RRad). Expression data are Log2 signal ratios values which were converted to colors according to the bar shown at the top: positive values correspond to genes over-expressed in isolated myofibers (red), whereas negative values refer to genes over-expressed in whole muscles (green), and therefore under-expressed in myofibers.</p>", "links"=>[], "tags"=>["genetics and genomics", "Computational biology"], "article_id"=>467938, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016807.g006", "stats"=>{"downloads"=>3, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Differential_expression_among_individual_fast_fibers_/467938", "title"=>"Differential expression among individual fast fibers.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-22 02:12:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/797327"], "description"=>"<p>Expression data are Log2 signal ratios values (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016807#pone.0016807.s003\" target=\"_blank\">Dataset S1</a>). The different color code emphasizes distinction of fiber types: positive values are in yellow and negative values in blue. Genes with differential expression between type 1 (soleus) and type 2B (EDL) myofibers were grouped according to functional classification: <i>i</i>) sarcomeric proteins (GO: contractile fiber part); <i>ii</i>) calcium signaling (GO: sarcoplasmic reticulum or calcium binding); iii) nucleus (GO: regulation of transcription or nucleus).</p>", "links"=>[], "tags"=>["signatures", "myofibers", "revealed", "two-class", "sam"], "article_id"=>467694, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016807.g004", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Molecular_signatures_of_fast_and_slow_myofibers_revealed_by_two_class_SAM_analysis_/467694", "title"=>"Molecular signatures of fast and slow myofibers revealed by two-class SAM analysis.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-22 02:08:14"}
  • {"files"=>["https://ndownloader.figshare.com/files/797713"], "description"=>"<p>Functional classification of 1,505 DE genes identified by two-class SAM analysis (FDR below 0.25%). GO enrichment was performed with the GOTM tool: general categories were identified, which are shown in bold letters and are associated to P-values (the lower, the better). Several sub-categories were further identified with the DAVID tool, which are associated to a score number (the higher, the better). Additional information in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016807#pone.0016807.s005\" target=\"_blank\">Dataset S3</a>.</p>", "links"=>[], "tags"=>["classification", "de", "genes", "two-class", "sam"], "article_id"=>468074, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016807.t002", "stats"=>{"downloads"=>2, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Functional_classification_of_DE_genes_identified_by_two_class_SAM_analysis_/468074", "title"=>"Functional classification of DE genes identified by two-class SAM analysis.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-02-22 02:14:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/797179"], "description"=>"<p><b>A</b>) Heat map of selected DE genes identified by one-class SAM analysis. Expression data are Log2 signal ratios values (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016807#pone.0016807.s003\" target=\"_blank\">Dataset S1</a>) which were converted to colors according to the bar shown at the top: positive values correspond to genes over-expressed in isolated myofibers (red), whereas negative values refer to genes over-expressed in whole muscles (green), and therefore under-expressed in myofibers. Mean values were calculated for two spot replicates. <b>B</b>) Validation by qPCR of four DE genes identified by one-class SAM analysis. Signal ratios (natural log values) were calculated independently in pools of 50 type 1 and 50 type 2B myofibers compared to the whole muscle control. The bars in the histogram correspond to the arithmetic mean of the two values separately calculated for type 1 and type 2B fibers. Normalization is relative to two internal references Mfn1 and Txn1; the vertical bars symbolize the intra-assay SD. Positive values correspond to genes over-expressed in myofibers (red bars), and negative values in whole muscles (green bars), as in the heat map.</p>", "links"=>[], "tags"=>["analyses", "allowed", "non-muscle", "cells", "enrichment"], "article_id"=>467541, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016807.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Single_fiber_analyses_allowed_removal_of_non_muscle_cells_and_enrichment_for_muscle_specific_genes_/467541", "title"=>"Single fiber analyses allowed removal of non-muscle cells and enrichment for muscle specific genes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-22 02:05:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/797457"], "description"=>"<p><b>A</b>) Discriminant genes identified by PAM tool. Expression data are Log2 signal ratios values (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016807#pone.0016807.s003\" target=\"_blank\">Dataset S1</a>). Positive values are in yellow color and negative values in blue color (according to the bar shown at the top). Results sorted by ranking were split in two parts, in order to show genes with preferential expression in type 1 (soleus) or type 2B (EDL) myofibers. <b>B</b>) Validation by qPCR of DE genes identified by PAM analysis. Signal ratios (natural log values) were calculated independently in pools of type 1 (gray bars) and type 2B (white bars) myofibers compared to whole muscle control. Normalization is relative to two internal references Mfn1 and Txn1; the vertical bars symbolize the intra-assay SD. Note that the expression of myostatin was not detectable in type 1 myofibers (*).</p>", "links"=>[], "tags"=>["discriminant", "emphasized"], "article_id"=>467830, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016807.g005", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_discriminant_analysis_emphasized_the_discovery_power_of_single_cell_analyses_/467830", "title"=>"The discriminant analysis emphasized the discovery power of single cell analyses.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-22 02:10:30"}
  • {"files"=>["https://ndownloader.figshare.com/files/796918"], "description"=>"<p><b>A</b>) Transmitted light images at 2.5X magnification of isolated muscle fibers from soleus (top) and EDL (bottom). Intact, unblemished myofibers appears as translucent cylinders. The inset shows details of the characteristic striated pattern (magnification 40X). Black scale bars: 250 µm; white scale bars: 25 µm. <b>B</b>) MyHC electrophoretic characterization of single fibers fragments from soleus (top) and EDL (below) muscles. A whole muscle sample has been used as marker of molecular weight (*). As shown in the examples, type 1 and type 2A fibers are abundant in the slow soleus muscle; type 2B and hybrid 2B/2X fibers are most frequent in the fast EDL muscle. <b>C</b>) Electropherogram of total RNA extracted from a single soleus myofiber, analyzed in the Agilent 2100 Bioanalyzer using a RNA 6000 Pico LabChip. About 1/3 of the full amount recovered was loaded in this experiment. The high quality of total RNA is confirmed by the presence of ribosomal peaks with no shift to lower fragments (RNA degradation) and no additional signals (DNA contamination).</p>", "links"=>[], "tags"=>["microgenomic", "technologies", "skeletal"], "article_id"=>467286, "categories"=>["Biological Sciences", "Genetics"], "users"=>["Francesco Chemello", "Camilla Bean", "Pasqua Cancellara", "Paolo Laveder", "Carlo Reggiani", "Gerolamo Lanfranchi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0016807.g001", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Experimental_set_up_of_microgenomic_technologies_in_skeletal_muscles_/467286", "title"=>"Experimental set up of microgenomic technologies in skeletal muscles.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-22 02:01:26"}

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