Urine exosome mRNA-based test for monitoring kidney allograft rejection: Effects of sample transportation and storage, and interference substances

Figure 1 Stability of exosomal mRNA upon urine transportation at different temperatures, and effects of various urine upfront processing techniques. A: Donor 1; B: Donor 2; C: Donor 3; D: Donor 4. For each donor, levels of 9 mRNA targets were analyzed by quantitative polymerase chain reaction; the graph displays Ct values (y-axis). Conditions mimicking urine transportation and handling, left to right: 2 d at +4C; 2 d at +20C; 2 d at +40C; freeze/thaw; 2x freeze/thaw; urine freeze/thaw without pre-processing.

Figure 2 Optimal refrigeration of urine specimens upon conditions mimicking shipment. One, two, three or four 12 oz gel packs were frozen overnight at -20C and placed in the 1.5 inch thick styrofoam box, to provide refrigeration for urine specimens. The graphs display temperature inside the box, over the range of 72 h.

Figure 3 Stability of exosomal mRNA upon prolonged urine storage at different temperatures. A: Donor 1; B: Donor 2; C: Donor 3; D: Donor 4. For each donor, levels of 9 mRNA targets were analyzed by quantitative polymerase chain reaction; the graph displays Ct values (y-axis). Urine storage conditions, left to right: 2 d at +4C; 7 d at +4C; 14 d at +4C; 4 d frozen at -80C; 30 d frozen at -80C.

Figure 4 Effect of common drugs on performance of the exosome molecular assay. Urine samples derived from 4 donors were utilized, and various drugs were spiked into urine before exosome purification, at levels exceeding the typical urinary concentrations by 50-fold. Nine mRNA targets were analyzed by quantitative polymerase chain reaction; the graph displays Ct values (y-axis). Drugs, left to right: Control, Tacrolimus, Mycophenolate, Cyclosporin A, Sirolimus, Everolimus, Teriflunomide, Ascomycin.

Figure 5 Effect of blood on performance of the exosome molecular assay. Urine samples derived from 4 donors were utilized, and blood or serum was spiked into urine before exosome purification. Levels of 9 mRNA targets were analyzed by quantitative polymerase chain reaction; the graph displays Ct values (y-axis). Conditions left to right: control urine specimens, processed following the standard protocol; urine specimens spiked with 1% blood and processed without additional centrifugation; urine specimens spiked with 1% blood and pre-processed following the standard protocol (centrifugation for 20 min at 2000 × g); urine specimens spiked with 1% serum and processed without additional centrifugation.