Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Stratagene human universal reference RNA labelled with different labelling protocols to test labelling protocols

ABSTRACT: Considerable variation in gene expression data from different DNA microarray platforms has been demonstrated. However, no characterization of the source of variation arising from labeling protocols has been performed. To analyze the variation associated with T7-based RNA amplification/labeling methods, aliquots of the Stratagene Human Universal Reference RNA were labeled using 3 eukaryotic target preparation methods and hybridized to a single array type (Affymetrix U95Av2). Variability was measured in yield and size distribution of labeled products, as well as in the gene expression results. All methods showed a shift in cRNA size distribution, when compared to un-amplified mRNA, with a significant increase in short transcripts for methods with long IVT reactions. Intra-method reproducibility showed correlation coefficients >0.99, while inter-method comparisons showed coefficients ranging from 0.94 to 0.98 and a nearly two-fold increase in coefficient of variation. Fold amplification for each method was positively correlated with the number of present genes. Two factors that introduced significant bias in gene expression data were observed: a) number of labeled nucleotides that introduces sequence dependent bias, and b) the length of the IVT reaction that introduces a transcript size dependent bias. This study provides evidence of amplification method dependent biases in gene expression data.

ORGANISM(S): Homo sapiens

SUBMITTER: Federico Alberto Monzon

PROVIDER: E-GEOD-3254 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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In vitro transcription amplification and labeling methods contribute to the variability of gene expression profiling with DNA microarrays.

Ma Changqing C Lyons-Weiler Maureen M Liang Wenjing W LaFramboise William W Gilbertson John R JR Becich Michael J MJ Monzon Federico A FA

The Journal of molecular diagnostics : JMD 20060501 2

The effect of different amplification and labeling methods on DNA microarray expression results has not been previously delineated. To analyze the variation associated with widely accepted T7-based RNA amplificationand labeling methods, aliquots of the Stratagene Human Universal Reference RNA were labeled using three eukaryotic target preparation methods followed by uniform replicate array hybridization (Affymetrix U95Av2). Method-dependent variability was observed in the yield and size distribu ...[more]

PMID: 16645204

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Project description:Acute lung rejection is a risk factor for chronic rejection, jeopardizing the long-term survival of lung transplant recipients. At present, acute rejection is diagnosed by transbronchial lung biopsies, which are invasive, expensive, and subject to significant sampling error. In this study, we sought to identify groups of genes whose collective expression in BAL cells best classifies acute rejection versus no-rejection. BAL samples were analyzed from 32 unique subjects whose concurrent histology showed acute rejection (n=14) or no rejection (n=18). Global BAL cell gene expression was measured using Affymetrix U133A microarrays. The nearest shrunken centroid method with 10-fold cross validation was used to define the classification model. 250 runs of the algorithm were performed to determine the range of misclassification error and the most influential genes in determining classifiers. The estimated overall misclassification rate was below 20%. Seven transcripts were present in every classifier and 52 transcripts were present in at least 70% of classifiers; these transcripts were notable for involvement with T-cell function, cytotoxic CD8 activity, and granulocyte degranulation. The proportions of both lymphocytes and neutrophils in BAL samples increased with increasing probability of acute rejection; this trend was more pronounced with neutrophils. We conclude that there is a prominent acute rejection-associated signature in BAL cells characterized by increased T-cell, CD8+ cytotoxic cell, and neutrophil gene expression; this is consistent with established mechanistic concepts of the acute rejection response. Experiment Overall Design: Lung transplant recipients surviving at least 30 days after transplantation at the University of Minnesota were eligible for enrollment in the study. The study was approved by the Universityâs Institutional Review Board, and all patients provided written informed consent. Subjects underwent scheduled surveillance (n = 22) and clinically indicated (decreased pulmonary function tests or new radiographic abnormalities) bronchoscopies (n = 10), in which 100-200 cc of sterile saline was instilled into a single anatomic location (right middle lobe or lingula), and recovered using gentle suction. 4-6 transbronchial biopsies (TBB) were obtained from the same subsegmental location as the BAL, and 4-6 additional TBB were obtained from the lower lobe of the same lung. After sending samples for clinical tests, approximately 50 ml of the recovered BAL effluent was immediately placed on ice. BAL cell counts and differentials were determined with a hemocytometer. Specimens from subjects with active bronchopulmonary infections (as determined by history, exam, chest radiograph, and the results of routine laboratory tests and cultures) were excluded from analysis. Experiment Overall Design: All biopsy specimens were graded according to standard International Society for Heart and Lung Transplantation criteria8. Vascular and airway cellular infiltrate were scored separately on A (vascular) and B (airway) scales, each score ranging from 0 to 48;23;24. For the purposes of this analysis, acute rejection was defined as a combined A+B score greater than or equal to 2; and no-rejection was defined as a combined A+B score less than 2. Experiment Overall Design: The BAL samples used in this study were selected according to strict criteria that were designed to maximize potential differences in gene expression between acute rejection and non-acute rejection BAL samples; and to minimize bias from confounding factors. Each subject (n=32) was represented by one BAL sample in this study. Control subjects and samples (n=14) were selected according to the following criteria: 1) A+B score was 0 or 1 for all biopsies collected from each subject during his/her post-transplant course; 2) at least three biopsies had been graded from each subject; 3) the subject had survived at least one year post-transplant; 4) the selected BAL sample was culture negative for pathogenic bacteria, fungi, and viruses; and 5) the selected sample was from a patient that had not developed BOS or was collected at least 6 months prior to the development of BOS. Rejection subjects and samples (n=18) were selected according to the following criteria: 1) the BAL sample was culture negative for pathogenic bacteria, fungi, and viruses; 2) for subjects with more than one acute rejection sample, we used the first acute rejection sample (A+B score > 1); 3) BOS grade was 0 at the time of BAL sampling (although some subjects subsequently developed BOS).

Dataset Information

Transcription profiling of Stratagene human universal reference RNA labelled with different labelling protocols to test labelling protocols

Publications

In vitro transcription amplification and labeling methods contribute to the variability of gene expression profiling with DNA microarrays.

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