Circulerende MicroRNA Kwantificering Met behulp van DNA-bindende Dye Chemistry and Droplet Digital PCR

The overall goal of this procedure is to quantify circulating microRNAs and biological fluids, such as plasma or serum, using droplet digital PCR technology. The method can help answer question in the biomarket field. By identifying diagnostic and prognostic, biomarketing cancer.

The main advantage of this technique is at the high sensitivity. It can be used to quantify also the less abundant microRNAs without requiring an androgynous reference gene. Implication of this method extend toward cancer diagnostic because it has the potential to identify people with early stage cancer.

It can also be applied to other diseases such as neurological or cardiac disorders. Generally, individuals need to pay attention to the manipulation of droplets before thermal cycling step to avoid their disruption. Hence, a visual demonstration of the droplet generation is critical because the droplets can be destroyed by careless manipulation or too fast pipetting.

Begin this procedure by isolating plasma or serum microRNAs. Then reverse transcribing and diluting them as described in the accompanying document. Remove the EvaGreen master mix microRNA primer set and cDNA from the freezer and allow them to thaw at room temperature.

After it has thawed, invert the master mix tube several times. And then spin down all of the reagents. Prepare a droplet digital, or ddPCR mix working solution according to the instructions in the accompanying document.

Prepare enough for up to eight samples with a no-template control for each condition plus an excess of 10%Once assembled, thoroughly mix and spin down the ddPCR mix. Note that technical replicates are not required due to the high-reproducibility of this technology. After the spin, dispense 12 microliters of ddPCR mix into each well of a 96-well PCR plate.

Add eight microliters of diluted cDNA template to each well. Then insert the droplet generator cartridge into the cartridge holder. To the middle row of the droplet generator cartridge, carefully transfer 20 microliters of each prepared sample.

Avoid introducing air bubbles to the bottom of the well. Fill each of the oil wells in the bottom row with 70 microliters of droplet generator oil. Hook the gasket over the cartridge holder.

Insert into the droplet generator and close the lid to start the droplet generation. When the droplet generation has finished, open the lid, remove the cartridge from the droplet generator, and remove the disposable gasket. The top wells of the cartridge contain the droplets.

Slowly and smoothly transfer 40 microliters from each of the eight top wells into a single column of a 96-well PCR plate. Immediately seal the plate with foil to avoid evaporation. Within 30 minutes of sealing the plate, begin thermal cycling using the PCR protocol described in table three of the accompanying document.

Power on the droplet reader. Then move the plate from the thermal cycler to the droplet reader. Place the 96-well PCR plate containing the post-PCR droplets into the base of the plate holder in the droplet reader.

Place the top of the plate holder on the PCR plate. Firmly press both release tabs down to secure the PCR plate in the holder. Start the QuantaLife software from the system PC.Click Setup and open the template data file which has information about the samples, target genes, and assays.

From the left-side navigation bar, select Run. A Run Options window will appear. From the Dye Set menu, select EVA, and click OK to start the droplet reading.

Once the run is complete, use the 2D Amplitude plot in the analysis software to select the positive droplets. To select the positive droplets, use the lasso tool from the left-side navigation bar. Next, click on the Events tab to check the number of positive and total droplets.

A total number of 18, 000 droplets are usually achieved with probeless ddPCR. Then click on the Concentration tab to visualize the final sample of concentration. Finally use the Export.

CSV to export the microRNA concentration values. The mRNA miR-181a-5p was quantified in two plasma samples, S1 and S2, using 0.5 and one microliter primer concentration at annealing temperatures of 58 and 60 degrees Celsius. This figure presents the copies of microRNA per microliter in the amplification reaction for each condition.

PCR positive droplets for each sample are shown in blue, and negative droplets are shown in black. As can be seen here, the control sample has no positive droplets as expected. Performing the PCR at 60 degrees Celsius with using either a 0.5 or one microliter of primer resulted in a better separation of positive and negative droplets.

In this figure, the error bars represent the Poisson 95%confidence interval. And here, the number of positive droplets shown in blue, are compared to the total number of droplets, shown in green. Taken together, these data demonstrate that the droplet digital PCR can be used to determine the absolute number of copies of microRNA per microliter of a biological fluid.

After watching this video, you should have a good understanding on how to perform a mRNA quantification using droplet digital PCR. Once mastered, 96 samples can be analyzed in just a few hours, which is an optimal output for a clinical setting. After each development, this technique pave the way to the field of liquid biopsy to explore microRNA and other nucleic acid as disease biomarkers.