In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Using species-specific primers, cpsDNA was quantified by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models.
Xenotransplantation is a feasible method to to treat However, effective monitoring of the immune rejection of xenotransplantation is a problem for physicians and the researchers. This protocol is a simple, convenient, low cost and less invasive method to monitor the immune rejection of the xenotransmutation. This technique can be used in xenotransplantation field, including pig-to-human islet transplantation, kidney transplantation, liver transplantation and heart transplantation.
Begin by transferring 500 microliters of blood samples into different micro centrifuge tubes. Add 20 microliters of protease K and 500 microliters of lysis buffer into the tubes. Then thoroughly shake them to mix.
Put the tubes in a water bath at 56 degrees Celsius for 10 minutes, shaking them two to three times during the incubation until the solution becomes clear centrifuge them briefly to remove the liquid beads from the inner wall of the tube covers. Then add 500 microliters of Anhydrous, Ethyl Alcohol, and shake thoroughly. Transfer the mixture into the adsorption column.
Then centrifuge the column for two minutes and discard the waste liquid. Add 800 microliters of rinse solution to each adsorption column, and centrifuge for one minute. leave the columns at room temperature for a few minutes to dry the remaining rinse solution.
Transfer the absorption columns to a clean centrifugal tube. Add 50 microliters of elution buffer to the middle of the adsorption films and place them at room temperature for two to five minutes, then centrifuge them for one minute. Prepare the PCR master mix according to manuscript directions, then add 23 microliters of the mix into each 0.6 milliliter micro centrifuge tube.
Add two microliters of genomic DNA to each tube and carefully Cabot. Then briefly mix and centrifuge. Place the tubes in a PCR cycler and start the amplification.
When the reaction is finished perform Agarose electrophoresis. Add 1.2 grams of Agarose into a flask containing a hundred millimeters of TAE and boil it for five minutes in the microwave. Once the Agarose has cooled to approximately 70 degrees Celsius, add five microliters of nucleic acid dye into the flask, slowly, pour it into the plate and leave it at room temperature until it solidifies into a gel.
Add five microliters of each sample and 2-Log DNA Ladder or DNA Marker 1, into the wells of the Agarose gel and electrophoresis at 120 million pier until the bands are separated. Visualize the gel with an ultraviolet imager. After performing transformation as described in the text manuscript, verify the plasmids by double restriction enzyme digestion with EcoR I and Bam HI.Separate the digested products with 1%Agarose electrophoresis and expose the gel to UV light.
Dilute the concentrated plasmid with the fragment of porcine DNA to two times 10 to the 11th copies per milliliter for the starting standard solution, Using double distilled water. To establish a standard curve, prepare a qPCR master mix, as described in the text manuscript and add 40 microliters of the mix into each tube of an eight-tube strip. Add 10 microliters of standard DNA of different concentrations, cap the tubes then briefly mix and centrifuge them.
Place the tubes in the qPCR machine and perform the amplification. Use EDTA tubes to collect blood samples of about 400 microliters from the pig-to-monkey artery patch models, or 100 microliters from the pig-to-mouse cell transplantation models. Then transfer the samples to 1.5 milliliter centrifuge tubes.
Remove the blood cells from the blood samples by centrifugation at low temperature and high speed. Transfer the supernatant to a new 1.5 milliliters centrifuge tube and remove the cell debris by centrifugation at 16, 000 times G for 10 minutes. Transfer the supernatant to a new 1.5 milliliter centrifuge tube and extract the circulating DNA using a commercial serum while circulating DNA extraction kit.
Condense the volume of circulating DNA to 40 microliters and store it at negative 20 degrees Celsius. Perform qPCR to quantify the circulating pig-specific DNA. Porcine-specific primers were designed and used to quantify circulating pig-specific DNA by qPCR, in pig-to-mouse cell transplantation models and pig-to-monkey artery patch, transplantation models.
Agarose electrophoresis was used to isolate amplified DNA fragments. Using PCR, the primers-specific for amplified porcine genomic DNA were identified. Next, species specificities of these primers in the cohort of monkey or human genomic DNA or in the cohort of mouse genomic DNA, were confirmed.
Finally two species-specific primers, were used to amplify pig DNA in the pig-to-monkey artery patch and pig to mouse, cell transplantation models. The most important thing to remember while attempting this protocol, is that contamination of all sorts should be prevented. This technique paves the way for researchers to explore new questions within xenotransplantation because it is a simple low-cost and non invasive method to monitor the immune rejection of xenotransplantation.
