Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Microarray analysis of mouse whole genome using self-amplifying messenger (SAM) RNA delivered with different formulations compared to controls

ABSTRACT: RNA-baffsed vaccines have recently emerged as a promising alternative to the use of DNA-based and viral vector vaccines, in part due to the potential to simplify how vaccines are made and facilitate a rapid response to newly emerging infections. SAM vaccines are based on engineered self-amplifying mRNA (SAM) replicons encoding an antigen, formulated with a synthetic delivery system, and induce broad-based immune responses in preclinical animal models. BALB/c mice (three biological replicates per group) were injected bilaterally intramuscularly (i.m.) with 50 Î¼l per quadriceps of Phosphate Buffered Saline alone (Control group), Ribonucleic Acid Expressing Respiratory Syncytial Virus F Protein (RNA) (500 ng), lipid nanoparticle (LNP) alone, or LNPâformulated RNA (LNP_RNA). A single quadriceps from each mouse was homogenized using QIAzol reagent and the FastPrepâ24 instrument (MP Bio). Total RNA was purified using RNeasy columns according to the manufacturerâs instructions (Qiagen). RNA labeling, hybridization, and scanning were performed using methods, reagents, software, and hardware purchased from Agilent Technologies. Briefly, 100 ng RNA was retro transcribed, labeled using Cy3, and column purified Qiagen. The efficiency of Cy3 dye incorporation was assessed on a NanoDrop 1000 instrument. Reactions with specific activities >6 pmol dye/Î¼g cRNA were used for hybridization. cRNA (1.65 Î¼g) was hybridized onto a 4 X 44 Whole Mouse Genome Microarray (G4122F). After scanning, images were analyzed using Feature Extraction 10.7.3.1 software. Microarray data was analyzed using the R/Bioconductor Limma openâsource package. Briefly, each spot was background corrected and spot intensities between arrays normalized using the quantile method. The average normalized spot intensity from experimental replicates was determined and differentially expressed genes (compared to the PBS control group) calculated using a moderated tâstatistic (Empirical Bayes method) followed by BenjaminiâHochberg false discovery rate (FDR) correction. Genes with an FDR adjusted pâvalue â¤ 0.05 and a foldâchange greater than or less than 2 (log2 = 1) were considered significantly changed. Functional clustering was performed using the DAVID online database. Heat maps were generated using Spotfire 4.0.3 software.

ORGANISM(S): Mus musculus

SUBMITTER: Timothy Pepini

PROVIDER: E-MTAB-5603 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Introduction: The mouse skeletal muscle is composed of four distinct fiber types that differ in contractile function, number of mitochondria and metabolism. Every muscle group has a specific composition and distribution of the four fiber types. Until now, three genes (CnA, PGC1α and PPARδ) are identified that are involved in the generation of more oxidative muscle types. In the present study we searched for novel genes that are involved in specifying different muscle types. Methods: Gastrocnemius and quadriceps muscles were dissected from 20-week old C57BL/6J mice. Gene expression profiles were compared at the level of the whole-transcriptome. The diet-sensitivity of genes differentially expressed between muscle types was explored by comparing mice fed a low fat diet with mice fed a high fat diet. Results: We identified 162 differentially expressed genes in the gastrocnemius as compared with the quadriceps. Genes with the strongest regulations were markers for oxidative fiber types, Hoxd8, Hoxd9 and Hoxd10 and others involved in embryogenesis. Additionally, diet did not influence the expression levels of genes specifying muscle types. Finally, when extrapolating our data to the soleus we could not find a corresponding gene expression pattern between Hoxd8, Hoxd9 and any of the fiber-type specific markers. However, based on these gene expression patterns we could distinguish the gastrocnemius, quadriceps and soleus from each other. Conclusion: Hoxd genes and genes that are markers for the different fiber types specify muscle type in a diet-independent way The aim of the present study was to find novel genes that may play a role in specifying muscle types. Therefore we compared gene expression profiles of the gastrocnemius with the quadriceps at the level of the whole-transcriptome. Functional implications were assessed by the analyses of predefined gene sets based on Gene Ontology, biochemical, metabolic and signaling pathways. muscle-type comparison

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Microarray analysis of mouse whole genome using self-amplifying messenger (SAM) RNA delivered with different formulations compared to controls

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