Team:Munich/Measurement
Measurement
Criteria
Quantitative Data:
Developing our platform, we strive to deliver reproducible results employing state-of –the-art quantitative methods, such as real-time PCR, using enough replicates and appropriate controls.
Absolute Values:
It is important to report values in units with a physical meaning to ensure reproducibility and comparability of our data. We therefore deliberately chose assays that allow us to convert our measured data into absolute values using standard curves. This ensures that we characterize our platform using comparable units, which scientists in the future can refer to.
Adequate Statistics:
Performing statistics on experimental data is an essential part of data analysis. We therefore ensured to include appropriate numbers of biological or technical replicates to evaluate our data with appropriate statistics using suitable statistical tests.
Appropriate Controls:
Control experiments necessary to ensure our protocols and assays are working properly were discussed with supervisors and critically evaluated. We made sure to always include negative controls for each measurement, and designed our constructs and assays to include process controls such as the leakage assay for HiBiT and no-RT controls for qPCR.
Sensitive Reporters:
Luciferase-based reporters were used because they have a longer linear measurement range compared to fluorescence-based methods. Using the HiBiT Assay, we are able to distinguish between leaked protein compared to successfully exported protein inside vesicles, which contributes to faithful result interpretation.
Standardized Methods:
We integrate commercially available assays (e.g. HiBiT) and common laboratory assays (e.g. qPCR) for a robust workflow. Due to that, our platform can be easily adjusted for different scientific approaches, making it easier for scientists to tailor it to their needs.
Reproducible Protocols and Plasmids for the Community
We established a set of protocols for efficient vesicle secretion and characterization. Additionally, all plasmid constructs are supplied in the registry. Do you want to cooperate with us? Contact us directly! (igem.munich@gmail.com)
Here are the most essential protocols for using ALiVE:
Incorporation of Standardized Methods
Implementing a split-luciferase assay to characterize vesicle secretion
Bioluminescent signal from transfected HEK293T cells and supernatant is measured with a sensitive split-luciferase assay - Promega's Nano-Glo® HiBiT Extracellular Detection System. For that, we have fused a HiBiT peptide on the C-terminus of our respective CD63 and Gag constructs. When HiBiT is paired with LgBiT, the nanoluc luciferase enzyme is reconstituted, resulting in a sensitive luminescent signal. Furthermore, a standard curve allows conversion of measured RFU values to absolute values. This assay is carried out 48 or 72h after transfection and enables us to confirm vesicle formation, trafficking and purification outside living cells.
A sensitive split-luciferase Nano-Glo® HiBiT Extracellular Detection System enables us to confirm vesicle formation and trafficking outside living cells.
Determining Vesicular Export and Assay Leakiness
Firstly, each sample is divided in three parts: cell content, supernatant (SN) lysed and supernatant (SN) unlysed. To achieve supernatant lysis, we treat samples with Triton™ X-100 detergent and expose them to 60°C for 15 min. The percentage of Triton™ X-100 used is the highest percentage to still reach lysis while not interfering with the assay. Lysis enables the access of LgBiT to HiBiT, which results in bioluminescence.
Figure 1: Graphical overview of the measurement and characterization regarding the vesicular export. The HiBiT fusions were the backbone in various applications either for sensitive luciferase-based assays or as RNA templates for qPCR. Our luciferase-based measurement was demonstrated by prooving vesicle secretion, exosome purification optimization, and RNA quantification.
After successful laboratory processing of our samples, data analysis is performed to determine relative export and leakiness of the system. Each measurement plate includes a calibration curve with Promega’s HiBiT Control protein diluted linearly, applied in duplicates for each concentration. Data analysis is started by calculating linear regression for each calibration curve. After that, raw data from each well is standardized to the molarity of 1 fmol HiBiT protein and up-scaled to the whole well volume. To assess export and leakage of the system, we perform the calculations indicated below. Total HiBiT signal is calculated as the sum of cellular content and lysed supernatant signal. Cell signal in absolute values shown on Fig. 1 corresponds to extrapolated cellular content data. Absolute supernatant values are calculated by subtracting extrapolated values from unlysed supernatant from corresponding lysed supernatant values.
qPCR Analysis
Precise quantitative analysis of vesicular content on the RNA level begins with RNA isolation and reverse transcription into cDNA. Based on fluorescent dye incorporation into cDNA with FLuc-specific primers, a real-time quantitative PCR assay is performed to measure levels of FLuc transcript present inside exosomes or VLPs. To ensure correct results interpretation, control samples without template and without added primers are included in the analysis. Previous to that, samples also include controls for transfection conditions – a control without RNA-binding protein (L7Ae) and with a mock plasmid (iRFP). A standard curve is also added. To control for gDNA contamination, GAPDH, a house-keeping gene, is used as a reference. Once we obtained results, the amount of FLuc mRNA was calculated using the ΔCt or standard curve method. As we only use 1-2% of isolated RNA for further processing, the results of ΔCt are multiplied accordingly to reach 100%. Then, values are scaled up to the volume of each well and corrected for cell confluency (70-80%). This process yields the amount of FLuc transcript present in a single cell.
With qPCR we are able to determine the amount of FLuc transcript present in a single cell.
Novel Purification Method for Exosomes
To establish a purification method for exosomes that does not rely on expensive kits or ultracentrifugation, we engineered the exosomal marker protein CD63 via incorporation of a polyhistidine affinity tag into the large extracellular loop. This modification not only allows us to separate exosomes from other extracellular vesicles but also facilitates the separation of RNA-loaded/engineered exosomes from endogenous exosomes, simplifying downstream analysis. On Fig. 3, we show that 35 % of the load were found in the flow-through, 15 % were found within the wash fractions and the remaining 50 % were eluted using high imidazole concentration. We performed this purification for HEK293T samples.
Data Evaluation with Experts
When developing a new project, experts in the field always play an important role in strategy evaluation, guidance and consultancy. Turning to them, we have been able to get a lot of feedback, which we have integrated accordingly throughout the course of several months.
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