Dataset Information

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.

SUBMITTER: Keslar KS

PROVIDER: S-EPMC3781926 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Multicenter evaluation of a standardized protocol for noninvasive gene expression profiling.

Keslar K S KS Lin M M Zmijewska A A AA Sigdel T K TK Tran T Q TQ Ma L L Bhasin M M Rao P P Ding R R Iklé D N DN Mannon R B RB Sarwal M M MM Strom T B TB Reed E F EF Heeger P S PS Suthanthiran M M Fairchild R L RL

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 20130701 7

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. Sample ...[more]

PMID: 23802725

Similar Datasets

Project description:Purpose: Determine if gene expression profiles in urine sediment could provide non-invasive candidate markers for painful bladder syndrome (PBS) with and/or without Hunner lesions. Materials and Methods: Fresh catheterized urine was collected and centrifuged from control (n = 5), lesion-free (n = 5), and Hunner lesion bearing (n = 3) patients. RNA was extracted from the pelleted material and quantified by gene expression microarray (Affymetrix Human Gene ST Array). Results: Three biologically likely hypotheses were tested: A) all three groups are distinct from one another; B) controls are distinct from both types of PBS patients combined, and C) Hunner lesion PBS patients are distinct from controls and non-Hunner-lesion PBS combined. For statistical parity an unlikely fourth hypothesis was included: non-Hunner-lesion PBS patients are distinct from controls and Hunner lesion PBS combined. Analyses supported selective upregulation of genes in the Hunner lesion PBS group (hypothesis C), and these were primarily associated with inflammatory function. This profile is similar to that reported in a prior microarray study of bladder biopsies in Hunner lesion PBS. Conclusions: Urine sediment gene expression from non-Hunner-lesion PBS patients lacked a clear difference from that of control subjects, while the array signatures from PBS patients with Hunner lesions showed a clear, primarily inflammatory, signature. This signature was highly similar to that seen in a prior microarray study of bladder biopsies. Thus, although sample sizes were small, this work suggests that gene expression in urine sediment may provide a non-invasive biomarker for Hunner lesion, but not non-Hunner lesion, PBS. Urine (40-100 ml) was immediately placed on ice, transported to the laboratory and centrifuged at -4C for 5 minutes. Pellets were washed twice with ice-cold phosphate-buffered saline, suspended in 0.8 ml TRIzol and RNA was extracted according to the manufacturer's instructions. RNA was stored at -80oC until all samples had been collected, then analyzed at our Microarray Core Facility using the Affymetrix GeneChip Whole Transcript (WT) Sense Target Labeling Assay protocol, added to Human Gene 1.0 ST array chips, and annotated with gene symbol and functional information (Affymetrix). Probe level data were produced by Partek GS (6.10) using the gcRMA output option. Probe set signal intensities < 4.2 were considered absent, and if > 10 chips rated a probe set absent, that probe set was not considered for further analysis. Additionally, probe sets that did not have a gene symbol-level annotation (e.g., were considered 'hypothetical', or 'expressed sequence tags') were not considered for further analysis.

Dataset Information

Multicenter evaluation of a standardized protocol for noninvasive gene expression profiling.

Publications

Multicenter evaluation of a standardized protocol for noninvasive gene expression profiling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets