This method may help answer key questions in the diagnostic field, such as whether micro RNAs are valid biomarkers in cardiovascular disease. Compared to quantitative real-time PCR, digital PCR exhibits superior technical qualities for quantifying microRNAs in the circulation. As the sample is divided into about 20, 000 droplets, no duplicate measurements are necessary, and no standard curve is needed for quantification.
Although this method may provide insight into quantification of microRNAs in cardiovascular patients, it may also be applied to other patients and diseases, as other microRNAs have shown promise as biomarkers in cancer progression, it may also be applied to oncological patients. Generally, individuals new to this method will struggle because the generated droplets are fragile and need to be handled with care. After isolating RNA from serum samples, continue with reverse transcription to obtain more stable complementary DNA for dPCR.
To begin, thaw the reverse transcription kit on ice. Then spin the enzymes and the primers down. Next, prepare the master mix as described in the text protocol.
Combine 10 microliters of the master mix with five microliters of extracted RNA in each well of a 96-well plate. Then centrifuge the plate at 2, 000xg at four degrees Celsius for two minutes. Proceed with the reverse transcription as detailed in the text protocol.
First, thaw the commercial dPCR mix, cDNA and PCR primers on ice and centrifuge immediately before use. Then prepare the master mix as described in the text protocol. Add 1.33 microliter volumes of reverse transcription product to 18.67 microliters volumes of the master mix and centrifuge briefly.
Using a pipet, transfer 20 microliters of the sample into each well of the medium row of an eight well cassette. Then pipet 70 microliters of droplet generation oil into the oil wells of the cassette. Next, place a gasket on top of the cassette and place it in the droplet generator.
Close the droplet generator and wait until the three indicator lights are solid green. Using a pipet, carefully transfer 40 microliters of the droplet-formed sample into separate wells of a 96-well plate. Then seal the plate with dPCR foil at 180 degrees Celsius for four seconds.
Place the sealed PCR plate in the cycler and follow the cycling instructions outlined in the text protocol. Remove the plate from the thermocycler and transfer it to the base of a plate holder. Then place the top of the plate holder onto the PCR plate.
Finally, start the commercial dPCR software. In this protocol, digital PCR was used to quantify microRNAs in the serum of patients with cardiovascular disease. Samples were taken from 16 patients before, eight hours after, and 16 hours after percutaneous intervention.
Patients with ST-elevation myocardial infarction showed a significant increase in miR-499 levels compared to patients with stable coronary artery disease. Once mastered, this technique can be done in about an hour for droplet formation of 96 samples if it is done correctly. While attempting this procedure, it is important to keep in mind to handle the droplets with care.
After watching this video, you should have a good understanding of how to perform digital PCR correctly, by correct droplet formation, cycling, reading, and analyzing.
