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.

Figure 1

Evaluation of urine RNA. Using reagents and samples prepared at site one, each laboratory isolated RNA from three urine pellet samples for qPCR analysis. A) Nanodrop absorbance spectra of 7 urine RNA samples isolated at site 1 illustrates typical appearance of urine RNA. B) Agilent Bioanalyzer electropherogram of a representative urine RNA indicates mostly small fragments of RNA; C) Nanodrop OD readings of the RNA isolated from urine pellet 1 in each site, showing normal yield and purity for urine samples; D) qPCR analysis of 3 urine pellet samples for 18S, and two samples for CXCL9, CXCL10, and IL8. All pre-analytical steps were performed at the respective sites using centrally provided reagents. Quantity and grand mean for each target and sample are plotted on a log scale. 18S sample 1 and CXCL10 samples 1 and 2 have one missing data point; all other samples show data from 5 sites.