Multicenter evaluation of a standardized protocol for noninvasive gene expression profiling

Abstract

Gene expression profiling of transplant recipient blood and urine can potentially be used to monitor graft function, but the multitude of protocols in use make sharing data and comparing results from different laboratories difficult. The goal of this study was to evaluate the performance of current methods of RNA isolation, reverse transcription, and quantitative polymerase chain reaction (qPCR) and to test whether multiple centers using a standardized protocol can obtain the same results. Samples, reagents, and detailed instructions were distributed to six participating sites that performed RNA isolation, reverse transcription and qPCR for 18S, PRF, GZB, IL8, CXCL9 and CXCL10 as instructed. All data were analyzed at a single site. All sites demonstrated proficiency in RNA isolation and qPCR analysis. Gene expression measurements for all targets and samples had correlations >0.938. The coefficient of variation of fold-changes between pairs of samples was less than 40%. All sites were able to accurately quantify a control sample of known concentration within a factor of 1.5. Collectively, we have formulated and validated detailed methods for measuring gene expression in blood and urine that can yield consistent results in multiple laboratories.

The Identification of Novel Potential Injury Mechanisms and Candidate Biomarkers in Renal Allograft Rejection by Quantitative Proteomics

“…Several reports on assessing mRNA level in urine sediments (12,37,38), urine protein (13,35) and peptides (16) have highlighted the feasibility of identifying potential biomarker molecules in urine. Measurement of urine mRNA levels presents an attractive biomarker option, but susceptibility of mRNA in the urine in combination with low mRNA abundances requires more validation in the clinical setting (39). Unlike peak clustering for SELDI-TOF or a label-free shotgun MS/MS approach, the quantitative iTRAQ method provides an accurate assessment of protein levels in the urine samples analyzed in this study.…”

Allograft rejection and tubulointerstitial fibrosis in human kidney allografts: Interrogation by urinary cell mRNA profiling

“…It is noteworthy that the multi-center CTOT-04 trial has not only validated the accuracy of urinary cell mRNA profiling for the detection of ACR in single center studies but also the utility of urinary cell mRNA profiles for the prediction of future occurrence of an ACR. It is also noteworthy that the reproducibility of the urinary cell mRNA protocols across laboratories has been examined and the overall, gene expression measurements had a correlation greater than 0.938 for the samples in this independent study [96]. …”

Analytical Aspects of the Implementation of Biomarkers in Clinical Transplantation

“…Its members have published standardized protocols to perform ELISPOT analyses of IFN-g generation by activated T cells, 12 gene expression profiling, 104 and multiplex analysis of HLA antibodies. 118 As mentioned above, the European ONE study consortium (http://www.onestudy.org/) has launched a standardization project of whole blood immune phenotype monitoring for clinical trials based on flow cytometry.…”