The overall goal of this procedure is to measure differentially methylated preproinsulin DNA species in human serum as a biomarker of beta cell death. This method can help answer key questions in the diabetes field, such as, is beta cell death occurring at a given time point during the development of type one diabetes? The main advantage of this technique is that by utilizing digital PCR, we can determine the amount of beta cell death in very small amounts of serum.
After collecting serum from human blood samples according to the text protocol, prepare 200 microleters of lysis buffer and one microleter of poly(A)per sample in a microcentrifuge tube. Vortex the tube for 15 seconds. Remove serum samples from the negative 80 degrees Celsius freezer and thaw them at room temperature.
Then, use PBS to bring the serum sample volume up to 200 microleters. Lyse the samples by adding 20 microleters of protease and 200 microleters of the lysis buffer poly(A)mix. Vortex the tubes for eight seconds and incubate the samples at 56 degrees Celsius for 10 minutes.
Then, centrifuge the tubes at top speed for seven seconds. Precipitate the DNA by adding 230 microleters of 100%ethanol to the samples and vortex the tubes for eight seconds. Then, centrifuge the tubes at top speed for seven seconds.
Next, apply 600 microleters of sample mixtures to spin columns and centrifuge the columns at 6000 times G for one minute. Discard the flowthrough and place the columns in clean tubes. Add 500 microleters of wash buffer one to the columns and centrifuge the samples at 6000 times G for one minute.
Then, discard the flowthrough and place the columns in clean tubes. Add 500 microleters of wash buffer two to the columns and centrifuge them at top speed for three minutes. Then, place the columns in new 1.5 millileter microcentrifuge tubes and spin them again for one minute.
To elute the DNA, place the columns in new 1.5 millileter microcentrifuge tubes and add 60 microleters of elution buffer directly to the filters. After incubating the columns at room temperature for five minutes, centrifuge the samples at 6000 times G for one minute. Discard the column and store the samples until ready for bisulfite conversion.
To convert unmethylated cytosines to uracils, add 130 microleters of bisulfite conversion reagent to 20 microleters of DNA in PCR tubes. Mix the samples 20 times by pipetting up and down and briefly spin the tubes to make sure no drops are on the sides or the lid. Incubate the samples in a water bath or thermal cycler as shown here.
Place the columns in the provided collection tubes and add 600 microleters of binding buffer to the spin columns. Apply the bisulfited DNA to the columns and mix by pipetting up and down 10 times. Then, centrifuge the samples at top speed for 30 seconds and discard the flowthrough.
Next, add 100 microleters of wash buffer to the column and centrifuge for 30 seconds. Then, to remove the sulfinate groups to finalize the conversion of unmethylated cytosines to uracil, add 200 microleters of desulfination buffer to the columns. After allowing the samples to stand at room temperature for 20 minutes, centrifuge the columns for 30 seconds.
Wash the DNA by adding 200 microleters of wash buffer to the columns and centrifuge them for 30 seconds. Then, discard the flowthrough and repeat the wash step. Place the columns into 1.5 millileter microcentrifuge tubes and elute the DNA by adding 10 microleters of elution buffer directly to the column filters.
Incubate the samples for one minute, then spin them for 30 seconds to elute DNA. Discard the columns and store the samples. Quantify the recovered DNA by spectophotometry at an absorbence of 260 nanometers.
Make a master mix with enough solution for each sample and control. Include at least one sample containing water, one sample of plasmid containing unmethylated insulin, and one sample containing a one to one mixture of plasmids. Set up multiplex PCR reactions in a 96 well plate by first adding 19.5 microleters of master mix to each well.
Add 2.5 microleters of bisulfite converted DNA sample into the appropriate wells. Then, mix the wells by pipetting up and down several times. Use foil and a plate sealer to seal the plate and centrifuge it in a plate spinner until there is no liquid on the sides of the wells.
To set up the automated droplet generator on the touchscreen set the number of rows being used by touching the rows in which samples are loaded in the wells of the plate. To avoid contamination, load the consumables from back to front, beginning with cartridges along the back row of the instrument. Then, load the tips into the center row.
Next, place the 96 well plate into the instrument. Then, after removing a cold block from the minus 20 degrees Celsius freezer, place a new skirted 96 well plate into it. Insert the block with the plate into the instrument, next to the plate with samples.
Add oil to the dispenser in front of the instrument and select the type of oil on the touchscreen. Touch the blue Start button to start the run. Then, confirm the plate setup and touch the Start Run button to begin.
Once the run is finished, remove the 96 well plate that contains the newly formed droplets and use a plate sealer to seal it with foil. Now, perform PCR in a thermal cycler using the following program. Place the plate on the droplet reader and set up the reader by clicking on the Setup tab.
Then, open a new template by clicking Template New. Use the well editor to set the parameters for each sample. Give each sample a unique name.
Set the experiment to Rare Event Detection and set the master mix to the mix used to prepare the reaction. Give Target 1 a name, such as Unmethylated, and set Target 1 as unknown, or U.Give Target 2 a name, such as Methylated, and set Target 2 as reference, or R.Click the Run tab to start the run and enter a file name. Then, in the Run Options window, select the detection chemistry.
To analyze the data, open the results in the Analyze tab and use the 2D plot based on positive controls to analyze the data. This figure shows the 2D scatter plots corresponding to droplets for plasmid controls, containing bisulfite converted, unmethylated insulin DNA, methylated insulin DNA, and a one to one mixture of the two plasmids. Note that in this panel there is a slight shift in the FAM positive droplets into the VIC channel, indicating the probe for the methylated insulin DNA.
Similarly, there is a very slight shift of the VIC positive droplets in the FAM channel, indicating crossreactivity of the probe for unmethylated DNA with methylated DNA. To demonstrate linearity of the primers, mixtures of the two plasmids across several orders of magnitude were used in the PCR reactions. As shown here, varying concentrations of one plasmid can be linearly detected in the presence of a constant amount of the second plasmid.
In this figure, samples from four individuals with new onset type one diabetes and four control samples without type one diabetes were analyzed. The data show that individuals with type one diabetes exhibit elevated levels of both unmethylated and methylated insulin DNA compared to controls, as previously shown. Once mastered, this technique can be done in eight hours for 30 samples if it is performed properly.
While attempting this procedure, it's important to remember to avoid contamination during all steps. After its development, this technique paved the way for researchers in the field of diabetes to explore the use of this technique as a biomarker for the development of type one diabetes. After watching this video you should have a good understanding of how to determine the amount of beta cell death by measuring unmethylated and methylated preproinsulin in cell free DNA from human serum.
Don't forget that working with DNA extraction buffers can be extremely hazardous and precautions, such as wearing gloves and a lab coat, and not using disinfecting agents that contain bleach, should always be taken while performing this procedure.
