Session Time: 5:30pm-6:30pm
Presentation Time: 5:30pm-6:30pm
Location: Exhibit Hall E
Background: The diagnosis of acute rejection in kidney transplantation is still standardized by an invasive renal biopsy. Noninvasive tests and biomarkers that can replace the invasive procedure for detection of rejection in transplantation is required for improving outcome of renal allograft. A previous study has suggested that the expression analysis of urinary-cell mRNA for the CD3ε chain, interferon-inducible protein 10 (IP-10), and 18S rRNA can cover invasive mean for diagnosing AR in US kidney transplant recipients. Recent study reported that droplet digital PCR (ddPCR) allowing higher sensitivity, reproducibility, and absolute quantification of gene expression without standard curves could be a very useful tool for various researches. We investigated whether these three genes are useful to diagnose AR in Korean renal transplant patients and compared ddPCR and qPCR in urine specimens.
Methods: 65 urine specimens (25 stable, 27 ACR, and 13 AMR) were collected after transplantation. RNA isolated from urine specimens was eluted in 30 ul nuclease-free water, and cDNA was synthesized with equal volume of 10 ul total RNA in all urines without quality control. The expression levels of transcripts for CD3ε, IP-10, and 18S rRNA were determined in urinary cells using qPCR and ddPCR and correlated transcript levels with renal allograft rejection.
Results: In the analysis of qPCR, CD3Ɛ and IP-10 were not detected, 30.8% and 35.4%, respectively; however, these genes were absolutely quantified in all samples by ddPCR. 18S ribosomal RNA (rRNA) was represented in 65 samples and all of these targets were increased in AMR patients by the methods. 18S rRNA-normalized CD3ε was significantly increased in patients with ACR compared with stable recipients in both methods, but ddPCR had greater statistical power and generated more significant result, p < 0.041 and p < 0.008. Increased level of 18S rRNA-normalized IP-10 was observed in ACR from ddPCR, and in AMR from qPCR.
Conclusion: From this study, only CD3ε among the three genes that were previously found as novel biomarkers for acute rejection was validated in Korean renal transplant patients in both qPCR and ddPCR assays. With more sensitive and accurate quantification, ddPCR could be a useful tool to monitor acute rejection by urine mRNA analysis after renal transplantation.
To cite this abstract in AMA style:Seo J-W, Moon H, Lee A, Choi Y-W, Kim Y, Moon J-Y, Lee S-H. A Noninvasive Diagnostic Test of Renal Allograft Rejection Using Urinary-Cell mRNA: A Comparison of Droplet Digital and Real-Time PCR [abstract]. Am J Transplant. 2015; 15 (suppl 3). https://atcmeetingabstracts.com/abstract/a-noninvasive-diagnostic-test-of-renal-allograft-rejection-using-urinary-cell-mrna-a-comparison-of-droplet-digital-and-real-time-pcr/. Accessed August 14, 2020.
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