Results

Experiment 1: ASO Protein Knockdown

Plate Setup and Translation:

We set up our sample translation key to map each ASO sequence and its technical replicate with a sample number for easier RT-qPCR well plate translation.

Table 1. Sample Translation.
Sample	Sequence ID	Sample	Sequence ID
1	F8-1	13	M-1
2	F8-2	14	M-2
3	F8-3	15	M-3
4	F10-1	16	L-1
5	F10-2	17	L-2
6	F10-3	18	L-3
7	F2-1	19	E7-1
8	F2-2	20	E7-2
9	F2-3	21	E7-3
10	E9-1	22	D11-1
11	E9-2	23	D11-2
12	E9-3	24	D11-3

RT-qPCR Plate Set up

Table 2. RT-qPCR Plate Set up. We set up our RT-qPCR plate depending on our multi-channel pipette for easy pipetting

We set up our RT-qPCR plate to include each corresponding stress granule protein with its corresponding ASO hypothesized to knock down. We had GAPDH as a control for all proteins, and also tested all proteins against the media and lipo control for a baseline comparison. We set up a primer efficiency curve against different concentrations of protein.

Raw CQ Data

After RT-qPCR, we obtained Quantification Cycle (Cq) values for each of our wells. Cq represents the cycle number at which the PCR reaction outputs a fluorescent signal, indicating the presence of the target effect.

Table 3. Snippet of raw CQ values. Each well in the 384 well plate had a corresponding CQ value used for further analysis.
Well	Fluor	Target	$C_t$
A1	SYBR	Unknown	17.4888418
A2	SYBR	Unknown	17.97278926
A3	SYBR	Unknown	17.35276733
A4	SYBR	Unknown	17.27687518
A5	SYBR	Unknown	16.47634628
A6	SYBR	Unknown	16.72385299
A7	SYBR	Unknown	37.57573508
A8	SYBR	Unknown	37.96026952
B1	SYBR	Unknown	24.66717837
B2	SYBR	Unknown	24.0139328
B3	SYBR	Unknown	24.25665287
B4	SYBR	Unknown	24.47493426
B5	SYBR	Unknown	23.97851527
B6	SYBR	Unknown	24.43964582
B7	SYBR	Unknown	38.33198103
B10	SYBR	Unknown	21.54228912
B11	SYBR	Unknown	21.15599149
B12	SYBR	Unknown	20.84109656
B13	SYBR	Unknown	21.03476051
C1	SYBR	Unknown	18.56623842
C2	SYBR	Unknown	18.89703446
C3	SYBR	Unknown	17.91677228
C4	SYBR	Unknown	18.68972263
C5	SYBR	Unknown	20.61733761
C6	SYBR	Unknown	18.67075024

Primer Efficiency Curve

We ran a primary efficiency curve to understand how efficiently a pair of primers amplifies a target sequence. To conduct this, we ran serial dilutions and each dilution is subject to RT-qPCR using the corresponding primers of interest. The resulting Cq values were plotted against the logarithm of the template concentration and the slope is used to calculate amplification efficiency. This is a critical step because suboptimal primers may lead to skewed quantification, so it is vital to take into account primer efficiency when calculating resulting values.

Table 4. RT-qPCR Ct values for DAZAP, GAPDH, FAM-labeled target across serial dilution series (1, 1:2, 1:4, 1:8, 1:16, 1:32) and no template control *NTC)

Before conducting any calculations, it is vital to analyze trends across targets. The overall trend is that the Cq values increase with dilution, which is as expected because lower template concentrations take more cycles to reach the fluorescence threshold. Outliers were crossed out to ensure that the standard deviations of technical replicates had low standard deviations. It was necessary to perform this step because technical replicates are expected to have similar values. If two technical replicates have a similar value and the third deviates from this value, we can conclude that the third is an outlier and remove the value to ensure consistent results. N/A values for NTC were expected as no primer was added to ensure low contamination. The standard deviation for most replicates was low, suggesting good reproducibility once outliers were omitted. Unexpectedly, the NTC values for GAPDH do have values, suggesting low=level contamination with DNA or cDNA into these wells. However our samples amplified much earlier than the NTC values (~15 - 20), so the actual sample signal is strongly above background. GAPDH is highly expressed, which makes primer-dimers or minor contamination more visible in NTC wells, but our experimental samples are clearly amplified as expected.

Table 5. Log(sample quantity) values for each dilution
Dilution	Log (Sample quantity)
1	0
1:2	-0.3010299957
1:4	-0.6020599913
1:8	-0.903089987
1:16	-1.204119983
1:32	-1.505149978

In preparation for our primer efficiency curve, we calculate log(sample quantity) for each dilution. In qPCR, our goal is to measure how the amount of DNA increases during each PCR cycle. The ideal is that the DNA doubles every cycle, which is exponential growth as shown in the following equation: $N = N_0(1+E)^C$ where N is the amount of product at cycle C, N₀ is the initial amount of template, and E is the primer efficiency. Logarithm is used in this case because exponential relationships are tricky to analyze, so to make it linear, we take the logarithm of the entire equation. The equation then becomes $\log(N) = \log(N_0) + C\log(1+E)$. The slope of this line is proportional to primer efficiency.

Figure 1 Primer efficiency curve: Average Cq Values vs. Log (Sample) for DAZAP1. Line equation: -3.98*x + 19.3 R^2 = 0.975

Figure 1 Primer efficiency curve. Average Ct vs Log(sample) for DAZAP1.

Figure 2 Primer efficiency curve: Average Cq Values vs. Log (Sample) for GAPDH. Line equation: -4.17*x + 15.5 R^2 = 0.992

Figure 2 Primer efficiency curve. Average Ct vs Log(sample) for GAPDH.

Figure 3 Primer efficiency curve: Average Cq Values vs. Log (Sample) for FAM. Line equation: -3.46*x + 22.5 R^2 = 0.94

Figure 3 Primer efficiency curve. Average Ct vs Log(sample) for FAM.

For all three primers, the plot of the Ct values against the logarithm of the initial template concentration produced a linear relationship as expected for efficient primers, evident in the $R^2$ values shown in the graphs.

Table 6. Efficiency calculations based on slope for DAZAP1, GAPDH, and FAM
Primer	Slope	Efficiency
DAZ	-3.977576677	78.40595984
GAP	-4.169012789	73.72604767
FAM	-3.463829212	94.40070548

Based on the slope, the efficiency was calculated with the following equation:

\[ \text{Efficiency (\%)} = \left(10^{-\frac{1}{\text{slope}}} - 1\right) \times 100 \]

In qPCR, the efficiency of the PCR reaction says how well the primers amplify the target DNA in each PCR cycle. A slope of -3.32 gives 100% efficiency. A very steep slope means the efficiency is less than 100%, and a more shallow slope says that the efficiency is greater than 100%. The efficiency equation above converts the slope into a percentage. While our efficiencies were low for **DAZAP1** and **GAPDH**, we took this limitation into account for our future calculations to measure expression fold change.

DAZAP1 - Pfaffl Method

Table 7 Pfaffl inputs — **Table 7.** Pfaffl Method inputs (raw Ct for DAZAP1 & GAPDH).

Table 8 Averages — **Table 8.** Averages of biological replicates for media & lipo controls.

Table 9 Delta Ct — **Table 9.** ΔCt values across conditions.

Table 10 RQ — **Table 10.** Relative quantity (RQ) of DAZAP1 and GAPDH.

Table 11 Relative expression — **Table 11.** Relative gene expression normalized to GAPDH.

Table 12 DeltaDeltaCt — **Table 12.** ΔΔCt & expression fold change.

Tables 7 - 11 show a stepwise analysis of DAZAP1 expression relative to GAPDH using qPCR. After all calculations and accounting for outliers, we see that the expression folder change is ~79 percent, sharing that DAZAP1 causes a 21 percent knockdown of the ASO of interest.

FAM - Delta Delta Ct Method

Table 13. Raw Ct Values for TDP-43 (FAM) and GAPDH Knockdown.

Table 12 has Ct values for FAM expression across three technical and biological replicates for treated, media only, and lipofectamine only samples. The treatment group has six samples (7-12), the media group has three (13-15), and the lipofectamine group has three (16-18). With three biological replicates, we were able to remove outliers between biological replicates to ensure our data points accurately described the true Ct value.

Table 14. Average and Standard Deviations of Biological Replicates.

FAM and GAPDH Ct values from biological replicates are compared across different experimental conditions. Average FAM Ct values for the treated samples are lower than that of media and lipo. In GAPDH, we see more grounded values across conditions, supporting its role as a reference gene. The standard deviations of the treated samples are 0 because only one biological replicate produced values outside the outlier range. While less than ideal, both treated technical replicates are similar in value across the two, supporting the Ct values for both.

Table 15. Delta CtE Values for FAM and GAPDH.

Table 16. Expression Fold Change.

While only one biological replicate produced accurate results in this experiment, this scenario is accounted for by the Delta Delta Ct method used, as shown in table 14. Expression fold changes show a 50–60% difference between the two technical replicates, showing that FAM has a 40–50% knockdown on the expression of the ASO of interest.

Experiment 2: Aptamer for TDP43 Mislocalization

Immunofluorescence single slice showing DAPI, G3BP1, and TDP-43 staining with sodium arsenite stress. — Single slice of TDP-43 antibody stain + G3BP1 antibody + DAPI + sodium arsenite stress (no stress granules observed).

Immunofluorescence single slice including aptamer staining on the 555 channel. — As left, with aptamer staining on 555.

Maximum intensity projection of Z-stack immunofluorescence with aptamer staining. — Maximum intensity projection of a Z-stack.

In the first image, which shows a single slice with TDP-43 antibody staining, G3BP1 antibody, DAPI, and sodium arsenite stress, the nuclei are clearly stained with DAPI and appear healthy and intact. The cytoplasmic staining, shown in red, appears faint and punctate in some areas but does not form large aggregates or stress granules. This matches the note that no stress granules were observed. Normally, sodium arsenite stress induces strong stress granule formation, but in this case the response was not visible, which may be due to insufficient stress conditions, variability in cell response, or technical issues with staining or imaging.

The second image includes the same staining as the first but adds aptamer staining on the 555 channel. The nuclei remain clearly stained, but the aptamer signal appears as scattered puncta with some background staining. There is weak cytoplasmic signal, possibly from G3BP1, but again there are no distinct stress granules. The scattered nature of the aptamer signal suggests that the staining may not yet be optimized and could be due to nonspecific binding or low target abundance.

The third image is a maximum intensity projection of a Z-stack with the same staining as the second image. The nuclei are again clearly visible, while the cytoplasmic staining is more distinct than in the previous images. The projection enhances weaker signals and allows for better visualization of small puncta. Some red and green puncta are more visible here compared to the single slice, but they still do not form the robust stress granules typically expected under sodium arsenite stress. The aptamer staining shows occasional puncta but does not co-localize strongly with stress granule markers.