Quantification of Circular RNAs Using Digital Droplet PCR

dd-PCR is a highly sensitive and accurate method for absolute quantification of target DNA or RNA. It has been used for disease diagnosis where other conventional methods fail to give accurate results. dd-PCR is the most sensitive technique for detecting very low abundant nucleic acid molecules.

Unlike quantitative PCR, dd-PCR doesn't require any housekeeping gene control for quantification. Due to its sensitivity and accuracy, dd-PCR can be used as a tool for molecular diagnostics and research involving low abundant RNA molecules like micro RNAs and circular RNAs. Before using dd-PCR, one should perform RT-PCR or RT-qPCR to confirm that the primers are amplifying their target genes without any non-specific PCR amplification.

To begin, prepare a PCR template sequence of 200 nucleotides in length by joining the last 100 nucleotides of the total circular RNA sequence length to the first 100 nucleotides of the circular RNA sequence. Use the template sequence for primer, designed by the Primer 3 web tool. Take one 10 centimeter dish containing about 5 million proliferating C2C12 myoblast cells and one four-day differentiated C2C12 myotube for RNA isolation.

Wash the cells with 10 milliliters of 1X PBS three times and discard the PBS using a pipette. Add one milliliter of RNA isolation reagent and lyse the cells by vigorous pipetting. Then, add 200 microliters of chloroform and vortex for 15 seconds.

Centrifuge the tube at four degrees Celsius for 10 minutes at 12, 000 G.Take 400 microliters of the upper aqueous layer, load it on a silica column, and centrifuge for one minute at 12, 000 G at room temperature. Take the flow through to a new tube and add 600 microliters of 100%ethanol. Add 20 microliters of magnetic silica beads and place the tube on a thermomixer, set at 25 degrees Celsius and 1, 200 RPM for five minutes.

Then, place the tube on a magnetic stand for 30 seconds or until the solution becomes clear. Resuspend the magnetic silica beads in 500 microliters of wash buffer containing 90%ethanol. After placing the tube on the magnetic stand for 30 seconds, let the beads settle toward the magnet and discard the buffer using a pipette.

Air dry the tube at 50 degrees Celsius for three minutes on a thermomixer, keeping the lid open. Add 20 microliters of nuclease free water and resuspend the beads. Collect the dissolved RNA in a new tube for quantity and quality assessment using a spectrophotometer.

Measure the RNA concentration using a spectrophotometer and take one microgram of RNA for cDNA synthesis. Mix one microgram of total RNA with one microliter of dNTP mix, four microliters of 5X reverse transcriptase buffer, two microliters of 10X reverse transcriptase random primer, 0.25 microliters of reverse transcriptase enzyme, and 0.5 microliters of RNase inhibitor. Make up the volume to 20 microliters using nuclease free water.

Place the tube at 25 degrees Celsius for 10 minutes, followed by 50 degrees Celsius for one hour. Then place the tube at 85 degrees Celsius for five minutes to inactivate the enzyme. Set up the PCR reaction of 22 microliters in 0.2 milliliter PCR tubes or strips, along with a negative control tube and non-template control tubes.

Add 11 microliters of dd-PCR master mix, 5.5 microliters of circular RNA-specific forward and reverse primer mix of one micromolar concentration, and 5.5 microliters of nuclease free water to set up the NTC reaction tubes. Similarly, to set up the test reaction tubes, add 11 microliters of dd-PCR master mix, 5.5 microliters of circular RNA specific forward and reverse primer mix of one micromolar concentration, and 5.5 microliters of cDNA. For the droplet generation, transfer 20 microliters of the PCR mixture from 0.2 milliliter PCR tubes to the sample wells of the droplet generator cartridge.

Add 70 microliters of droplet generation oil to the oil wells of the droplet generator cartridge carefully, using a pipette. Cover the droplet generator cartridge with a rubber gasket and place it in the droplet generator machine to get the sample oil droplet mix, generated in the droplet wells of the cartridge. For PCR amplification, transfer 40 microliters of the sample oil droplet mix from the droplet wells of the droplet generator cartridge to a 96-well PCR plate using a pipette.

Seal the 96-well PCR plate with the aluminum foil sealer. Place it in the plate sealer machine block, preheated at 180 degrees Celsius, and click seal for sealing the PCR plate. Then, place the plate in the dd-PCR thermal cycler and set the lid temperature to 105 degrees Celsius and a ramp rate to two degrees Celsius per second.

Once PCR is over, place the plate on the dd-PCR droplet counter machine with the plate holder in the correct position for counting the droplets. Open the droplet analysis software and set up the run by giving input of the sample information. Click the analyze button to analyze the data after the completion of the droplet reading.

Then, click the 1D Amplitude button to see the positive and negative droplets. To segregate the positive droplets from the negative droplets, put a common threshold line for all samples with the same target. Click the export button to export the circular RNA counts data as a csv file.

The exported data show the number of circular RNAs present in each sample. Manually calculate the number of circular RNAs in each sample per nanogram of RNA by considering the total amount of cDNA used in each reaction. The data analysis using QuantaSoft Software is shown here.

All samples except MT1 showed more than 12, 000 droplet counts. Since the total droplet count was low in MT1, this sample was not considered for the final data analysis. Interestingly, there was a clear difference in the expression pattern of Circ B and C2 in the four day differentiated myotube condition compared to the myoblast cells.

The analysis of Circ B and C2 abundance in terms of copy number, indicated that the three replicates of C2C12 myoblasts had 76.8, 67, and 46 copies per nanogram of RNA, while the two myotube samples had 558 and 610 copies per nanogram of RNA. The data in the bar graph represent the mean plus minus standard deviation of two to three biological replicates. ddPCR is one of the most advanced method to measure the accurate differential expression of target circular RNA.

This method will be an essential tool to accelerate circular RNA research and diagnostic industries in the coming year.