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Expression of inflammatory and structural matrix genes in synovial fluid following intra-articular administration of isoflupredone acetate to exercised horses

ABSTRACT: In the current study, we use gene expression approach to assess the effects and duration of action of IPA on the expression levels of genes encoding pro and anti-inflammatory genes and structural proteins in an exercised horse model.Equine specific microarrays containing expression profiling of 25,923 genes were used. Baseline samples collected the day prior to IPA administration were compared to those collected at 24 hours and on day 7 and samples from day 7 compared to 24 hours. The volcano plots of differentially expressed abundant transcripts generated from microarray analysis are depicted in Figure 2. Comparison of baseline samples to 24 hours revealed 855 differentially expressed genes with 632 genes up-regulated and 223 genes down regulated. Comparison of baseline samples to those collected on day 7 showed 23,358 genes differentially expressed with 23,318 genes up-regulated and 40 down regulated. When samples from day 7 were compared to 24 hours, 26,411 genes were differentially expressed with 26,409 genes up-regulated and 2 genes down regulated. Based on the results of the microarray analysis (greatest fold change and statistical significance), 20 genes were chosen for further analysis to quantitate the change in expression levels, relative to baseline, at several time points post drug administration.

ORGANISM(S): Equus caballus

PROVIDER: GSE98410 | GEO | 2017/05/03

SECONDARY ACCESSION(S): PRJNA385182

REPOSITORIES: GEO

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Project description:Background: The transcriptome of peripheral white blood cells (PWBCs) contains valuable physiological information, thus making them a prime biological sample for investigating mRNA-based biomarkers. However, prolonged storage of whole blood samples can alter gene transcript abundance in PWBCs, compromising the results of biomarker discovery. Here, we designed an experiment to interrogate the impacts of delayed processing of whole blood samples on gene transcript abundance in PWBCs. We hypothesized that storing blood samples for 24 hours at 4°C would cause RNA degradation resulting in altered transcriptome profiles. Results: We produced RNA-sequencing data for 30 samples collected from five estrus synchronized heifers (Bos taurus). We quantified transcript abundance for 12,414 protein-coding genes in PWBCs. Analysis of parameters of RNA quality revealed no statistically significant differences (P>0.05) between samples collected from the jugular vein and coccygeal vein, as well as among samples processed after 1, 3, 6, or 8 hours. However, samples processed after 24 hours of storage had a significantly lower RNA integrity number value (P=0.03) in comparison to those processed after one hour of storage. Next, we analyzed RNA-sequencing data between samples using those processed after one hour of storage as the baseline for comparison. Interestingly, evaluation of 3’/5’ bias revealed no statistically significant differences between genes with lower transcript abundance in samples stored for 24 hours relative to one hour. In addition, sequencing coverage of transcripts was similar between samples from the 24 hours and one-hour groups. We identified four and 515 genes with differential transcript abundance in samples processed after storage for eight and 24 hours, respectively, relative to samples processed after one hour. Conclusions: The PWBCs respond to prolonged cold storage by increasing genes related to active chromatin compaction which in turn reduces gene transcription. This alteration in transcriptome profiles can impair accuracy of mRNA-based biomarkers. Therefore, blood samples collected for mRNA-based biomarker discovery should be refrigerated immediately and processed within six hours post sampling.

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Expression of inflammatory and structural matrix genes in synovial fluid following intra-articular administration of isoflupredone acetate to exercised horses

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