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 μl 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 LNP–formulated RNA (LNP_RNA). A single quadriceps from each mouse was homogenized using QIAzol reagent and the FastPrep–24 instrument (MP Bio). Total RNA was purified using RNeasy columns according to the manufacturer’s 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/μg cRNA were used for hybridization. cRNA (1.65 μg) 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 open–source 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 t–statistic (Empirical Bayes method) followed by Benjamini–Hochberg false discovery rate (FDR) correction. Genes with an FDR adjusted p–value ≤ 0.05 and a fold–change 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