Sequential analysis of myocardial gene expression with phenotypic change: Use of cross-platform concordance to strengthen biologic relevance.
ABSTRACT: OBJECTIVES:To investigate the biologic relevance of cross-platform concordant changes in gene expression in intact human failing/hypertrophied ventricular myocardium undergoing reverse remodeling. BACKGROUND:Information is lacking on genes and networks involved in remodeled human LVs, and in the associated investigative best practices. METHODS:We measured mRNA expression in ventricular septal endomyocardial biopsies from 47 idiopathic dilated cardiomyopathy patients, at baseline and after 3-12 months of ?-blocker treatment to effect left ventricular (LV) reverse remodeling as measured by ejection fraction (LVEF). Cross-platform gene expression change concordance was investigated in reverse remodeling Responders (R) and Nonresponders (NR) using 3 platforms (RT-qPCR, microarray, and RNA-Seq) and two cohorts (All 47 subjects (A-S) and a 12 patient "Super-Responder" (S-R) subset of A-S). RESULTS:For 50 prespecified candidate genes, in A-S mRNA expression 2 platform concordance (CcpT), but not single platform change, was directly related to reverse remodeling, indicating CcpT has biologic significance. Candidate genes yielded a CcpT (PCR/microarray) of 62% for Responder vs. Nonresponder (R/NR) change from baseline analysis in A-S, and ranged from 38% to 100% in S-R for PCR/microarray/RNA-Seq 2 platform comparisons. Global gene CcpT measured by microarray/RNA-Seq was less than for candidate genes, in S-R R/NR 17.5% vs. 38% (P = 0.036). For S-R global gene expression changes, both cross-cohort concordance (CccT) and CcpT yielded markedly greater values for an R/NR vs. an R-only analysis (by 22 fold for CccT and 7 fold for CcpT). Pathway analysis of concordant global changes for R/NR in S-R revealed signals for downregulation of multiple phosphoinositide canonical pathways, plus expected evidence of a ?1-adrenergic receptor gene network including enhanced Ca2+ signaling. CONCLUSIONS:Two-platform concordant change in candidate gene expression is associated with LV biologic effects, and global expression concordant changes are best identified in an R/NR design that can yield novel information.
Project description:Benzene is an established cause of leukemia, and possibly lymphoma, in humans, but the underlying molecular pathways remain largely undetermined. We used two microarray platforms to identify global gene expression changes associated with well-characterized occupational benzene exposure in the peripheral blood mononuclear cells (PBMC) of a population of shoe-factory workers. Differential expression of 2692 genes (Affymetrix) and 1828 genes (Illumina) was found and the concordance was 50% (based on an average fold-change > or =1.3 from the two platforms), with similar expression ratios among the concordant genes. Four genes (CXCL16, ZNF331, JUN and PF4), which we previously identified by microarray and confirmed by real-time PCR, were among the top 100 genes identified by both platforms in the current study. Gene ontology analysis showed overrepresentation of genes involved in apoptosis among the concordant genes while pathway analysis identified pathways related to lipid metabolism. The two-platform approach allows for robust changes in the PBMC transcriptome of benzene-exposed individuals to be identified.
Project description:Microarray technologies are widely used to quantify the abundance of transcripts corresponding to thousands of genes. To maximise the robustness of transcriptome results, we have tested the performance and reproducibility of rat and mouse gene expression data obtained with Affymetrix, Illumina and Operon platforms.We present a thorough analysis of the degree of reproducibility provided by analysing the transcriptomic profile of the same animals of several experimental groups under different popular microarray technologies in different tissues. Concordant results from inter- and intra-platform comparisons were maximised by testing many popular computational methods for generating fold changes and significances and by only considering oligonucleotides giving high expression levels. The choice of Affymetrix signal extraction technique was shown to have the greatest effect on the concordance across platforms. In both species, when choosing optimal methods, the agreement between data generated on the Affymetrix and Illumina was excellent; this was verified using qRT-PCR on a selection of genes present on all platforms.This study provides an extensive assessment of analytical methods best suited for processing data from different microarray technologies and can assist integration of technologically different gene expression datasets in biological systems.
Project description:A DNA microarray platform, based on the nucleotide sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the rRNA gene, was developed to identify 32 fungal pathogens at the species level. The probe sequences were spotted onto polycarbonate slides with a mini-microarray printer, and after the hybridization, the results were visible with the naked eye. The performance of the microarray platform was evaluated against the commercial automated systems (Vitek 2 and BD Phoenix systems) and DNA sequencing (gold standard). A total of 461 blood culture bottles were tested: 127 positive for fungi, 302 positive for bacteria, and 32 that were negative. Once the microorganisms were identified by automated systems, fungal DNA was extracted directly from the blood culture bottles. The DNA products were tested using the microarray platform, and DNA sequencing was performed. The results of the microarray and DNA sequencing were concordant in 96.7% of cases, and the results from the automated systems and DNA sequencing were concordant in 98.4%. Of all the nucleotide sequences contained in the microarray platform, the microarray failed to identify four fungal isolates (one Candida parapsilosis, two Candida tropicalis, and one Cryptococcus neoformans). Of note, the microarray detected Candida krusei DNA in two blood cultures from the same patient, whereas the automated system was only positive for Enterococcus faecium Our microarray system provided reliable and fast fungal identification compared to that from DNA sequencing and the automated systems. The simplicity of reading the results by the naked eye made this DNA platform a suitable method for fungal molecular diagnosis.
Project description:Importance:Assessment of PD-L1 (programmed cell death 1 ligand 1) expression by immunohistochemical analysis has been used as a predictive diagnostic test to identify responders and guide treatment in trials of the PD-1 (programmed cell death 1) axis inhibitors. The definition of PD-L1 positive lacks standardization, and prediction of response by immunohistochemical analysis is additionally limited by the subjective nature of this technique. Objective:To examine whether PD-L1 antibody reagents are interchangeable by quantitatively comparing the expression of the PD-L1 protein. Design, Setting, and Participants:In this immunohistochemistry standardization study, 30 randomly selected cases of lung cancer resected from January 1, 2008, through December 31, 2009, were obtained from Yale Pathology Archives with a range of expression of PD-L1. To test for protein measurement, rather than clinical utility, a PD-L1 index tissue microarray, including cell line and tissue controls, was used. The results were then validated on a commercially available, genetically defined PD-L1 engineered cell line array with a range of controlled protein-expressing cell lines using 6 monoclonal antibodies (SP142, E1L3N, 9A11, SP263, 22c3, and 28-8). Protein levels were measured by quantitative immunofluorescence and quantitative chromogenic assessment. Data analysis was performed from September 2015 through May 2016. Results:Concordance between 4 antibodies revealed regression for tumor tissue cores (R2?=?0.42-0.91) and cell line cores (R2?=?0.83-0.97) by quantitative immunofluorescence in the PD-L1 index tissue microarray. All 6 antibodies had high levels of concordance (R2?=?0.76-0.99) when using chromogenic staining in isogenic cell lines. Conclusions and Relevance:Because the antibodies are highly concordant, these results suggest that assays based on the use of these antibodies could yield concordant results. They further suggest that previously described differences in PD-L1 expression in tissue are independent of the antibody used and likely attributable to tumor heterogeneity, assay- or platform-specific variables, or other factors.
Project description:In large-scale genome-wide association studies based on high-density single nucleotide polymorphism (SNP) genotyping array, the quantity and quality of available genomic DNA (gDNA) is a practical problem. We examined the feasibility of using the Multiple Displacement Amplification (MDA) method of whole-genome amplification (WGA) for such a platform. The Affymetrix Early Access Mendel Nsp 250K GeneChip was used for genotyping 224,940 SNPs per sample for 28 DNA samples. We compared the call concordance using 14 gDNA samples and their corresponding 14 WGA samples. The overall mean genotype call rates in gDNA and the corresponding WGA samples were comparable at 97.07% [95% confidence interval (CI), 96.17-97.97] versus 97.77% (95% CI, 97.26-98.28; P = 0.154), respectively. Reproducibility of the platform, calculated as concordance in duplicate samples, was 99.45%. Overall genotypes for 97.74% (95% CI, 97.03-98.44) of SNPs were concordant between gDNA and WGA samples. When the analysis was restricted to well-performing SNPs (successful genotyping in gDNA and WGA in >90% of samples), 99.11% (95% CI, 98.80-99.42) of the SNPs, on average, were concordant, and overall a SNP showed a discordant call in 0.92% (95% CI, 0.90-0.94) of paired samples. In a pair of gDNA and WGA DNA, similar concordance was reproducible on Illumina's Infinium 610 Quad platform as well. Although copy number analysis revealed a total of seven small telomeric regions in six chromosomes with loss of copy number, the estimated genome representation was 99.29%. In conclusion, our study confirms that high-density oligonucleotide array-based genotyping can yield reproducible data and MDA-WGA DNA products can be effectively used for genome-wide SNP genotyping analysis.
Project description:Risk assessment and treatment choice remains a challenge in early non-small-cell lung cancer (NSCLC). The aim of this study was to identify novel genes involved in the risk of early relapse (ER) compared to no relapse (NR) in resected lung adenocarcinoma (AD) patients using a combination of high throughput technology and computational analysis. We identified 18 patients (n.13 NR and n.5 ER) with stage I AD. Frozen samples of patients in ER, NR and corresponding normal lung (NL) were subjected to Microarray technology and quantitative-PCR (Q-PCR). A gene network computational analysis was performed to select predictive genes. An independent set of 79 ADs stage I samples was used to validate selected genes by Q-PCR.From microarray analysis we selected 50 genes, using the fold change ratio of ER versus NR. They were validated both in pool and individually in patient samples (ER and NR) by Q-PCR. Fourteen increased and 25 decreased genes showed a concordance between two methods. They were used to perform a computational gene network analysis that identified 4 increased (HOXA10, CLCA2, AKR1B10, FABP3) and 6 decreased (SCGB1A1, PGC, TFF1, PSCA, SPRR1B and PRSS1) genes. Moreover, in an independent dataset of ADs samples, we showed that both high FABP3 expression and low SCGB1A1 expression was associated with a worse disease-free survival (DFS).Our results indicate that it is possible to define, through gene expression and computational analysis, a characteristic gene profiling of patients with an increased risk of relapse that may become a tool for patient selection for adjuvant therapy.
Project description:BACKGROUND: We compared the relative precision and accuracy of expression measurements obtained from three different state-of-the-art commercial short and long-oligonucleotide microarray platforms (Affymetrix GeneChip, GE Healthcare CodeLink and Agilent Technologies). The design of the comparison was chosen to judge each platform in the context of a multi-project program. RESULTS: All wet-lab experiments and raw data acquisitions were performed independently by each commercial platform. Intra-platform reproducibility was assessed using measurements from all available targets. Inter-platform comparisons of relative signal intensities were based on a common and non-redundant set of roughly 3,400 targets chosen for their unique correspondence toward a single transcript. Despite many examples of strong similarities we found several areas of discrepancy between the different platforms. CONCLUSION: We found a higher level of reproducibility from one-color based microarrays (Affymetrix and CodeLink) compared to the two-color arrays from Agilent. Overall, Affymetrix data had a slightly higher level of concordance with sample-matched real-time quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR) data particularly for detecting small changes in gene expression levels.
Project description:RNAseq and microarray methods are frequently used to measure gene expression level. While similar in purpose, there are fundamental differences between the two technologies. Here, we present the largest comparative study between microarray and RNAseq methods to date using The Cancer Genome Atlas (TCGA) data. We found high correlations between expression data obtained from the Affymetrix one-channel microarray and RNAseq (Spearman correlations coefficients of ?0.8). We also observed that the low abundance genes had poorer correlations between microarray and RNAseq data than high abundance genes. As expected, due to measurement and normalization differences, Agilent two-channel microarray and RNAseq data were poorly correlated (Spearman correlations coefficients of only ?0.2). By examining the differentially expressed genes between tumor and normal samples we observed reasonable concordance in directionality between Agilent two-channel microarray and RNAseq data, although a small group of genes were found to have expression changes reported in opposite directions using these two technologies. Overall, RNAseq produces comparable results to microarray technologies in term of expression profiling. The RNAseq normalization methods RPKM and RSEM produce similar results on the gene level and reasonably concordant results on the exon level. Longer exons tended to have better concordance between the two normalization methods than shorter exons.
Project description:<h4>Motivation</h4>We have proposed a mixture model based approach to the concordant integrative analysis of multiple large-scale two-sample expression datasets. Since the mixture model is based on the transformed differential expression test P-values (z-scores), it is generally applicable to the expression data generated by either microarray or RNA-seq platforms. The mixture model is simple with three normal distribution components for each dataset to represent down-regulation, up-regulation and no differential expression. However, when the number of datasets increases, the model parameter space increases exponentially due to the component combination from different datasets.<h4>Results</h4>In this study, motivated by the well-known generalized estimating equations (GEEs) for longitudinal data analysis, we focus on the concordant components and assume that the proportions of non-concordant components follow a special structure. We discuss the exchangeable, multiset coefficient and autoregressive structures for model reduction, and their related expectation-maximization (EM) algorithms. Then, the parameter space is linear with the number of datasets. In our previous study, we have applied the general mixture model to three microarray datasets for lung cancer studies. We show that more gene sets (or pathways) can be detected by the reduced mixture model with the exchangeable structure. Furthermore, we show that more genes can also be detected by the reduced model. The Cancer Genome Atlas (TCGA) data have been increasingly collected. The advantage of incorporating the concordance feature has also been clearly demonstrated based on TCGA RNA sequencing data for studying two closely related types of cancer.<h4>Availability and implementation</h4>Additional results are included in a supplemental file. Computer program R-functions are freely available at http://home.gwu.edu/?ylai/research/Concordance.<h4>Contact</h4>email@example.com.<h4>Supplementary information</h4>Supplementary data are available at Bioinformatics online.
Project description:Endotracheal intubation and prolonged immobilization of patients receiving mechanical ventilation may reduce expectoration function. High-frequency chest wall oscillation (HFCWO) may ameliorate airway secretion movement; however, the instantaneous changes in patients' cardiopulmonary responses are unknown. Moreover, HFCWO may influence ventilator settings by the vigorous oscillation. The aim of this study was to investigate these issues.Seventy-three patients (52 men) aged 71.5?±?13.4 years who were intubated with mechanical ventilation for pneumonic respiratory failure were recruited and randomly classified into 2 groups (HFCWO group, n?=?36; and control group who received conventional chest physical therapy (CCPT, n?=?37). HFCWO was applied with a fixed protocol, whereas CCPT was conducted using standard protocols. Both groups received sputum suction after the procedure. Changes in ventilator settings and the subjects' responses were measured at preset intervals and compared within groups and between groups.Oscillation did not affect the ventilator settings (all P?>?0.05). The mean airway pressure, breathing frequency, and rapid shallow breathing index increased, and the tidal volume and SpO2 decreased (all P?<?0.05). After sputum suction, the peak airway pressure (Ppeak) and minute ventilation decreased (all P?<?0.05). The HFCWO group had a lower tidal volume and SpO2 at the end of oscillation, and lower Ppeak and tidal volume after sputum suction than the CCPT group.HFCWO affects breathing pattern and SpO2 but not ventilator settings, whereas CCPT maintains a steadier condition. After sputum suction, HFCWO slightly improved Ppeak compared to CCPT, suggesting that the study extends the indications of HFCWO for these patients in intensive care unit. (ClinicalTrials.gov number NCT02758106, retrospectively registered.).