Impact of delayed processing of bovine peripheral blood on differential gene expression
2012
Sheridan, Michael P. | Browne, John A. | MacHugh, David E. | Costello, Eamon | Gormley, Eamonn
RT-qPCR can be used to accurately determine expression levels of genes following RNA extraction from tissue samples. If blood is the source of total RNA, it is often desirable to process the samples immediately following collection because delays in processing for RNA extraction may influence mRNA expression estimates obtained from RT-qPCR analyses. However, this may not be feasible if the site of blood collection is distant from the processing laboratory. In the present study, the effects of delays in the processing of blood samples on mRNA expression data was investigated using a panel of 23 functionally diverse genes from five different gene ontology (GO) categories in peripheral blood sampled from ten age-matched healthy cattle. Venous blood was collected in Tempus™ Blood RNA tubes, which contain reagents that lyse blood cells immediately and stabilise the RNA signature (T₀). Blood was also collected in conventional lithium heparin collection tubes, and stored at ambient temperature for T₄, T₆ and T₈h, prior to total RNA extraction. The mRNA expression profiles of these 23 genes were determined by RT-qPCR and compared across the time course. Thirteen genes showed significant up- or down-fold changes in mRNA expression over the 8h time course. Among the GO categories, genes in the Immune response category showed the most differential expression. These results also demonstrated that the changes in mRNA expression for the IFNG gene, which encodes the cytokine IFN-γ, did not correspond to IFN-γ protein levels estimated using ELISA.
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