Transfection, which is the ability to modify host cells’ genetic content, has broad application in studying normal cellular processes, molecular mechanism of disease and gene therapy. There are several transfection techniques, and all require either a control or a reference gene. Commonly used controls for transfection experiments are housekeeping genes, which maintain expression for a given cell/tissue, experimental conditions, and treatment. However, recent research has uncovered that expression levels of housekeeping genes may vary depending on the gene, cell type and experimental conditions. A growing body of evidence demonstrates that housekeeping genes are inadequate internal standards for measuring gene expression levels as they are affected by many factors, including experimental treatment and conditions. Current literature is lacking about adequacy of tubulins, specifically β-tubulin isotypes as controls for transfection experiments. This research aims to fill this gap by testing whether tubulin is a suitable control gene for transfection in RAW264.7 cells. It is hypothesized that tubulin expression changes following transfection because tubulins play a role in genetic material uptake. RAW264.7 cells were transfected by electroporation with either non-targeting siRNA (NT-siRNA) or cyclophilin A siRNA (CyPA siRNA). Levels of β-tubulin isotypes mRNA was quantified by RT-qPCR for cells collect either 24 hours or 48 hours after transfection. Increased expression of βI (+50%, p<0.01) was observed 24 hours after electroporation with NT-siRNA. βI(-50%, p<0.001), βII(-50%, p<0.01) and βIII (-92%, p<0.01), βIV (-55%, p<0.001) expression decreased 24-hours post-electroporation (0.5±0.2, p<0.00124hours post-electroporation with CyPA. All four β tubulin isotypes were deemed unusable reference gene following transfection with electroporation involving CyPA targeting siRNA, specifically βIII tubulin would not be suitable control for transfection with electroporation due to these causing significant changes in expression levels, 24h post-transfection.
Chabra, Ankita, "Feasibility of Tubulin as a Control for Gene Expression following Transfection in Mouse Monocyte/Macrophage-like Cells" (2022). St. Mary's University Honors Theses and Projects. 16.
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