Implementation

One challenge was confirming whether lipotransfection would be effective in our experiment. When attempting to knock down stress granule proteins using ASOs, we faced challenges finding cell-type specific data about SH-SY5Y cells on transfection efficiency, making it difficult to optimize our protocol. As a result, we proceeded using generalized lipofectamine protocols. This lack of assurance that this method would work in our cell line added uncertainty to our results, as it may have reduced knockdown efficiency and highlights the need for future validation with SH-SY5Y-specific optimization or alternative delivery methods.

Another obstacle emerged during primer validation for RT-qPCR. In order to quantify transcript knockdown, we relied on RT-qPCR measurements normalized to a housekeeping gene. However, the results from the qPCR showed that it was taking less cycles than expected to reach the expected expression for the controls compared to the ASO-affected genes. We were expecting it to take at least 30 cycles, so that there would be a significant difference between the controls and the experimental factors to ensure that the ASOs did affect stress granule formation genes. However, our measured values for the controls came up to only about 28 cycles, while the ASOs varied from 20-24 cycles, a difference that was present but not as prominently as we expected. This was also seen when we tested our primers through standard curves. The calculated efficiencies were not as high or consistent as we had expected. For example, DAZ and GAPDH primer sets fell below the ideal 90–110% efficiency range, with values closer to ~78% and ~74%, respectively. Although FAM primers performed better (~94%), these discrepancies may have impacted the precision of our gene expression fold change calculations. Despite these challenges, the efficiency tests confirmed that our primers were functional, but highlighted the need for future optimization to strengthen the reliability of our qPCR measurements.

We also encountered difficulties when attempting to visualize the binding of our novel aptamer to the protein TDP-43 in stress granules, using fluorescent microscopy to obtain these images. The aptamer did not appear to label TDP-43, as it wasn't clear where the aptamer was binding to. This suggested either a limitation in the staining protocol we used, or a lack of direct binding between the aptamer and the protein. To troubleshoot, we repeated the protocol with adjustments to improve labeling. While we were not able to confirm precise binding to TDP-43, we did observe aptamer localization within the nucleus of stress granule–containing cells. This finding demonstrated that the aptamer successfully associated with stress granule–forming cells, but also highlighted the need for further optimization and alternative validation strategies to pinpoint its exact binding target.