The overall goal of this method is to assess the integrity of urine cell-free DNA at specific oncogene sequences. This method can help answer key questions in the urological cancer field, such as early non-invasive diagnosis of bladder and prostate cancer. The main advantage of this technique is that it is easy to do and to analyze.
The implications of this technique extend towards diagnosis of urological malignancies, because they normally release cells and free nucleic acids in urine. Though this method can provide insight into urinary samples, it can also be applied to other systems such as serum or plasma. Generally, individuals new to this method would struggle because the concentration of urine cell-free DNA is often low.
We first had the idea for this method when we tried to find a simple method for non-invasive early detection of bladder cancer. To begin the procedure, obtain a 50 milliliter clean catch first morning urine sample that has been stored at four degrees Celsius for no more than three hours. Invert the sample twice and then partition the sample into two 50 milliliter conical centrifuge tubes.
Centrifuge the sample at 850 times G for 10 minutes at four degrees Celsius. Then, for each tube transfer 10 milliliters of the upper part of the supernatant to two five milliliter tubes, leaving at least two milliliters of supernatant above the pellet. Discard the pellets and store the supernatant at minus 80 degrees Celsius.
Repeat this process for additional urine samples as needed. To begin DNA purification, for each sample thaw an aliquot of urine supernatant. Vortex the thawed sample and invert the tube twice.
Place one milliliter of the sample into a five milliliter tube, and store the remainder at minus 80 degrees Celsius. Prepare a DNA purification kit suitable for urine samples according to the manufacturer's instructions. Add 100 microliters of proteinase K and one milliliter of lysis buffer to the sample and mix well with a pipette.
Incubate the sample at 56 degrees Celsius for 15 minutes. During incubation, prepare the wash buffers in a spin column. After incubation, allow the sample to cool to room temperature and then add one milliliter of absolute ethanol and mix well by pipetting.
Transfer 650 microliters of the sample to the column. Centrifuge at 6000 times G for one minute. Replace the collection tube and repeat this process until all of the sample has been loaded onto the column.
Then add 500 microliters of the first wash buffer and centrifuge the column at 6000 times G for one minute. Replace the collection tube, add 500 microliters of the second wash buffer, and centrifuge the column at max speed for three minutes. Place the column in a clean collection tube and centrifuge at max speed for another three minutes to remove residual wash buffer.
Transfer the column to a clean 1.5 milliliter micro centrifuge tube. Load 50 microliters of elution buffer onto the column and allow the buffer to soak into the column for seven minutes. Then centrifuge the column at 6000 times G for one minute.
Pipette the eluate back into the column and centrifuge the column at max speed for one minute to obtain the urine cell-free DNA. To begin preparing the standards, first isolate DNA from an appropriate cell line using a purification kit. Quantify the DNA from each purified urine sample in the cell line with a spectrophotometer per the kit manufacturer's instructions.
Dilute the UCF DNA to one nanogram per microliter and store the diluted samples at minus 20 degrees Celsius until ready for the integrity test. Dilute the cell line DNA to create six, 100 microliter standards of different concentrations. Store the standards at minus 20 degrees Celsius.
To begin the DNA integrity test, thaw the UCF DNA samples, the standards, the appropriate PCR primers, and green supermix on ice. Then set up the 72-well rotor disc plate with strip tubes. Place two, 10 microliter aliquots of standard and then each sample into wells.
Prepare two wells of 10 microliter RNase-free water as negative controls. For each filled well, prepare a mix of one microliter of each primer, 12.5 microliters of green supermix, and 6.5 microliters of RNase-free water. Transfer 15 microliters of the mix into each filled well, and then transfer the wells into a 72-well rotor disc.
Perform the PCR with a realtime instrument according to the manufacturer's specifications. Using the instrument software, check if any replicates have a cycling threshold value difference of one or more. Discard those samples from the analysis and evaluate the median cycling threshold for the remaining samples.
Perform melting analysis on each sample with a median cycling threshold value of 36 or less to evaluate the PCR product specificity. Then use the standard curve to determine a concentration in nanograms per microliter for each applicon. Urine cell-free DNA was isolated and the DNA integrity was evaluated in four regions that show increased DNA copy number in solid tumors.
A control sequence was also evaluated to verify that the DNA quantity in each sample was sufficient for the integrity test. The total free DNA concentration and the coefficients of variation were calculated for each gene. This method shows 73%sensitivity and 84%specificity for detecting bladder cancer, but only 58%sensitivity and 44%specificity for prostate cancer.
Once mastered, this technique can be done in one working day if it is performed properly. Following this procedure, other methods like digital PCR or next generation sequencing can be performed in order to answer additional questions like copy number inducer of the genes of interest. After watching this video you should have a good understanding of how to isolate cell free DNA from urine in a very simple manner, and how to study the integrity of specific regions of interest using real time PCR approaches.
