Measurement
Precise and accurate measurements ensure that our results are reliable and validate the quality of our work. Using universal and standard measurements allows for our experiments to generate data that can be used as reference or for comparison by other groups. By using standardized approaches, we created a framework of measurements that can serve as a reference for both ALS researchers and the broader iGEM community. Our project required assessing two main aspects of ALS-related biology: changes in gene expression of stress granule proteins, and the molecular interactions of TDP-43 with RNA aptamers, including visualization of their effects on aggregation dynamics. To quantify the gene expression of proteins essential to stress granule formation and TDP-43 aggregation, we used rt-qPCR. We measured the efficacy of our TDP-43 aptamer through fluorescence imaging, binding assays, and protein interaction analyses. This ensured that our results were both reproducible and meaningful. These measurements allowed us to evaluate the efficiency of RNA aptamers in preventing harmful TDP-43 phase separation. Ultimately, our measurements formed the foundation for translating our findings into a potential therapeutic intervention and contributed to a broader understanding of neurodegenerative mechanisms.
Gene Expression Analysis
The purpose of our measurement was to determine the change in gene expression when cells were knocked down for stress granule proteins under stressful conditions to determine the role of these proteins in forming stress granules.
We treated SH-SY5Y cells with ASOs to knock down stress granule proteins (DAZAP1, FAM98A, and SND1). We checked for reduced transcript expression of these proteins using RT-qPCR.
Experimental Protocol
ASO Treatment
Cells were treated with antisense oligonucleotides (ASOs) at a final concentration of 5 µM. To prepare the transfection mix, 100 µL OPTI-MEM was combined with 1 µL Lipofectamine 3000 and 7.5 µL ASO per 300 µL of mix, incubated for 10 minutes, and 100 µL was added per well.
RNA Extraction & Quantification
RNA was extracted from treated cells and quantified using a Nanodrop spectrophotometer to assess concentration and purity (260/280 and 230/280 ratios). cDNA was synthesized from RNA using the ProtoScript® II First Strand cDNA Synthesis Kit.
qPCR Setup
qPCR reactions were prepared with a total volume of 10 µL per well: 4.5 µL ddH2O + cDNA (diluted to 8 ng/µL), 5.0 µL SYBR Green master mix, and 0.5 µL primer (0.25 µL forward, 0.25 µL reverse). Master mixes were calculated to include excess volume for pipetting error. All conditions were conducted with three technical replicates. qPCR plates were thermocycled (25°C for 5 min, 42°C for 1 hr, 12°C hold) and run according to standard amplification conditions. Data were collected for downstream analysis.
Pfaffl Method Analysis
With the raw results from our qPCR, we calculated the relative gene expression to calculate the expression fold change for proteins DAZ and FAM, using GAPDH as a reference. Data analysis followed the Pfaffl method, which accounts for primer efficiency when calculating relative expression. Our results confirmed efficient knockdown of stress granule proteins, validating the upstream step of our project.
Pfaffl Formula
This gives the relative gene expression ratio normalized to the housekeeping gene and relative to the reference.
Analysis Procedure
- Step 1: Determine primer efficiencies for genes of interest (DAZ and FAM) and housekeeping gene (GAPDH)
- Step 2: Convert % efficiency into exponential values (ex. 100% → 2.0)
- Step 3: Average technical replicates: Calculate the mean Ct values for DAZ, FAM, and GAPDH from replicate qPCR runs
- Step 4: Set our controls (media only, lipo only) as a reference point
- Step 5: For each sample, calculate the ∆Ct values by subtracting the reference CT value from the sample Ct value
- Step 6: Insert primer efficiencies and ∆Ct values into the Pfaffl formula
Primer Efficiency Validation
To confirm the validity of our results, we created a primer efficiency curve for each primer set by preparing serial dilutions of cDNA (8 ng → 0.25 ng) in three replicates and testing across primer sets. Based on our calculations, we discovered that as the primer gets more diluted in the serial dilutions, the efficiency drops as expected, which led us to believe that our primers were working effectively.
Primer Performance Results
GAPDH Primer
DAZ Primer
FAM Primer
We measured that GAPDH had a primer efficiency of 73.726, DAZ had a primer efficiency of 78.406, and FAM had the highest primer efficiency at 94.401.
Calculation Procedure for PCR primer efficiency
Primer Efficiency Formula
Standard Curve Visualizations
DAZ Standard Curve
Figure 6: Slope of standard curve for DAZ
GAPDH Standard Curve
Figure 7: Slope of standard curve for GAPDH
FAM Standard Curve
Figure 8: Slope of standard curve for FAM
Fluorescence Imaging
While qPCR allowed us to measure changes in transcript levels, fluorescence microscopy provided spatial and functional information about how aptamers interacted with TDP-43 in live cells. This step was essential because phase separation and aggregation are inherently dynamic processes that cannot be captured solely by gene expression assays. We checked for the aptamer, G3BP1, protein involved in stress granule formation and usually binds to TDP-43, and the nucleus, DAPI.
Microscopy Results
Imaging was conducted using fluorescence microscopy.
Aptamer Channel
G3BP1 Channel
DAPI Channel
Merged
Figure 9: Confocal fluorescence microscopy of the aptamer, G3BP1, and DAPI, and merged
Key Findings
Based on these images, it was apparent that the aptamer was found in the nucleus of the stress granule cells. However, there was vague overlap between the aptamer and G3BP1, which led us to believe that the aptamer may have not strongly bound to TDP43. Despite this, we were able to confirm that the aptamer was binding to a protein in the stress granule, highlighting that our designed sequences possessed sufficient structural complexity to bind with the intended targets. This highlighted the use of aptamers as selective molecular tools to modulate stress granule dynamics, and laid the foundation for further characterization of binding affinity, specificity, and potential functional consequences on TDP-43 aggregation.
Conclusion
Precise and accurate measurements ensure that our results are reliable and validate the quality of our work. Using universal and standard measurements allows for our experiments to generate data that can be used as reference or for comparison by other groups. By using standardized approaches, we created a framework of measurements that can serve as a reference for both ALS researchers and the broader iGEM community. Our project required assessing two main aspects of ALS-related biology: changes in gene expression of stress granule proteins, and the molecular interactions of TDP-43 with RNA aptamers, including visualization of their effects on aggregation dynamics. To quantify the gene expression of proteins essential to stress granule formation and TDP-43 aggregation, we used rt-qPCR. We measured the efficacy of our TDP-43 aptamer through fluorescence imaging, binding assays, and protein interaction analyses. This ensured that our results were both reproducible and meaningful. These measurements allowed us to evaluate the efficiency of RNA aptamers in preventing harmful TDP-43 phase separation. Ultimately, our measurements formed the foundation for translating our findings into a potential therapeutic intervention and contributed to a broader understanding of neurodegenerative mechanisms.