Experimental Comparison and Evaluation of the Affymetrix Exon and U133Plus2 GeneChip Arrays

Affymetrix exon arrays offer scientists the only solution for exon-level expression profiling at the whole-genome scale on a single array. These arrays feature a new chip design with no mismatch probes and a radically new random primed protocol to generate sense DNA targets along the entire length of the transcript. In addition to these changes, a limited number of validating experiments and virtually no experimental data to rigorously address the comparability of all-exon arrays with conventional 3'-arrays result in a natural reluctance to replace conventional expression arrays with the new all-exon platform.Using commercially available Affymetrix arrays, we assess the performance of the Human Exon 1.0 ST (HuEx) and U133 Plus 2.0 (U133Plus2) platforms directly through a series of 'spike-in' hybridizations containing 25 transcripts in the presence of a fixed eukaryotic background. Specifically, we compare the measures of expression for HuEx and U133Plus2 arrays to evaluate the precision of these measures as well as the specificity and sensitivity of the measures' ability to detect differential expression.This study presents an experimental comparison and systematic cross-validation of Affymetrix exon arrays and establishes high comparability of expression changes and probe performance characteristics between Affymetrix conventional and exon arrays. In addition, this study offers a reliable benchmark data set for the comparison of competing exon expression measures, the selection of methods suitable for mapping exon array measures to the wealth of previously generated microarray data, as well as the development of more advanced methods for exon- and transcript-level expression summarization

Accurate Estimates of Microarray Target Concentration from a Simple Sequence-Independent Langmuir Model

Background: Microarray technology is a commonly used tool for assessing global gene expression. Many models for estimation of target concentration based on observed microarray signal have been proposed, but, in general, these models have been complex and platform-dependent. Principal Findings: We introduce a universal Langmuir model for estimation of absolute target concentration from microarray experiments. We find that this sequence-independent model, characterized by only three free parameters, yields excellent predictions for four microarray platforms, including Affymetrix, Agilent, Illumina and a custom-printed microarray. The model also accurately predicts concentration for the MAQC data sets. This approach significantly reduces the computational complexity of quantitative target concentration estimates. Conclusions: Using a simple form of the Langmuir isotherm model, with a minimum of parameters and assumptions, and without explicit modeling of individual probe properties, we were able to recover absolute transcript concentrations with high R 2 on four different array platforms. The results obtained here suggest that with a ‘‘spiked-in’ ’ concentration serie

Implementation of exon arrays: alternative splicing during T-cell proliferation as determined by whole genome analysis

<p>Abstract</p> <p>Background</p> <p>The contribution of alternative splicing and isoform expression to cellular response is emerging as an area of considerable interest, and the newly developed exon arrays allow for systematic study of these processes. We use this pilot study to report on the feasibility of exon array implementation looking to replace the 3' <it>in vitro </it>transcription expression arrays in our laboratory.</p> <p>One of the most widely studied models of cellular response is T-cell activation from exogenous stimulation. Microarray studies have contributed to our understanding of key pathways activated during T-cell stimulation. We use this system to examine whole genome transcription and alternate exon usage events that are regulated during lymphocyte proliferation in an attempt to evaluate the exon arrays.</p> <p>Results</p> <p>Peripheral blood mononuclear cells form healthy donors were activated using phytohemagglutinin, IL2 and ionomycin and harvested at 5 points over a 7 day period. Flow cytometry measured cell cycle events and the Affymetrix exon array platform was used to identify the gene expression and alternate exon usage changes. Gene expression changes were noted in a total of 2105 transcripts, and alternate exon usage identified in 472 transcript clusters. There was an overlap of 263 transcripts which showed both differential expression and alternate exon usage over time. Gene ontology enrichment analysis showed a broader range of biological changes in biological processes for the differentially expressed genes, which include cell cycle, cell division, cell proliferation, chromosome segregation, cell death, component organization and biogenesis and metabolic process ontologies. The alternate exon usage ontological enrichments are in metabolism and component organization and biogenesis. We focus on alternate exon usage changes in the transcripts of the spliceosome complex. The real-time PCR validation rates were 86% for transcript expression and 71% for alternate exon usage.</p> <p>Conclusions</p> <p>This study illustrates that the Exon array technology has the potential to provide information on both transcript expression and isoform usage, with very little increase in expense.</p

Validation of Endogenous Control Genes for Gene Expression Studies on Human Ocular Surface Epithelium

PURPOSE: To evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. METHOD: The lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. RESULTS: The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB<18S<TBP<B2M<PGK1<HPRT1<GUSB<GAPDH<PPIA-RPLP0. In this study, geNorm analysis has shown the following ECGs pairs to be most stably expressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable. CONCLUSION: This study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use of PPIA-RPLP0 ECGs pair in quantitative reverse transcription PCR across the OS epithelium

Application of Equilibrium Models of Solution Hybridization to Microarray Design and Analysis

Background: The probe percent bound value, calculated using multi-state equilibrium models of solution hybridization, is shown to be useful in understanding the hybridization behavior of microarray probes having 50 nucleotides, with and without mismatches. These longer oligonucleotides are in widespread use on microarrays, but there are few controlled studies of their interactions with mismatched targets compared to 25-mer based platforms. Principal Findings: 50-mer oligonucleotides with centrally placed single, double and triple mismatches were spotted on an array. Over a range of target concentrations it was possible to discriminate binding to perfect matches and mismatches, and the type of mismatch could be predicted accurately in the concentration midrange (100 pM to 200 pM) using solution hybridization modeling methods. These results have implications for microarray design, optimization and analysis methods. Conclusions: Our results highlight the importance of incorporating biophysical factors in both the design and the analysis of microarrays. Use of the probe ‘‘percent bound’ ’ value predicted by equilibrium models of hybridization is confirmed to be important for predicting and interpreting the behavior of long oligonucleotide arrays, as has been shown for shor

Gene Expression Profiles from Formalin Fixed Paraffin Embedded Breast Cancer Tissue Are Largely Comparable to Fresh Frozen Matched Tissue

BACKGROUND AND METHODS: Formalin Fixed Paraffin Embedded (FFPE) samples represent a valuable resource for cancer research. However, the discovery and development of new cancer biomarkers often requires fresh frozen (FF) samples. Recently, the Whole Genome (WG) DASL (cDNA-mediated Annealing, Selection, extension and Ligation) assay was specifically developed to profile FFPE tissue. However, a thorough comparison of data generated from FFPE RNA and Fresh Frozen (FF) RNA using this platform is lacking. To this end we profiled, in duplicate, 20 FFPE tissues and 20 matched FF tissues and evaluated the concordance of the DASL results from FFPE and matched FF material. METHODOLOGY AND PRINCIPAL FINDINGS: We show that after proper normalization, all FFPE and FF pairs exhibit a high level of similarity (Pearson correlation >0.7), significantly larger than the similarity between non-paired samples. Interestingly, the probes showing the highest correlation had a higher percentage G/C content and were enriched for cell cycle genes. Predictions of gene expression signatures developed on frozen material (Intrinsic subtype, Genomic Grade Index, 70 gene signature) showed a high level of concordance between FFPE and FF matched pairs. Interestingly, predictions based on a 60 gene DASL list (best match with the 70 gene signature) showed very high concordance with the MammaPrint® results. CONCLUSIONS AND SIGNIFICANCE: We demonstrate that data generated from FFPE material with the DASL assay, if properly processed, are comparable to data extracted from the FF counterpart. Specifically, gene expression profiles for a known set of prognostic genes for a specific disease are highly comparable between two conditions. This opens up the possibility of using both FFPE and FF material in gene expressions analyses, leading to a vast increase in the potential resources available for cancer research

Unsupervised assessment of microarray data quality using a Gaussian mixture model

<p>Abstract</p> <p>Background</p> <p>Quality assessment of microarray data is an important and often challenging aspect of gene expression analysis. This task frequently involves the examination of a variety of summary statistics and diagnostic plots. The interpretation of these diagnostics is often subjective, and generally requires careful expert scrutiny.</p> <p>Results</p> <p>We show how an unsupervised classification technique based on the Expectation-Maximization (EM) algorithm and the naïve Bayes model can be used to automate microarray quality assessment. The method is flexible and can be easily adapted to accommodate alternate quality statistics and platforms. We evaluate our approach using Affymetrix 3' gene expression and exon arrays and compare the performance of this method to a similar supervised approach.</p> <p>Conclusion</p> <p>This research illustrates the efficacy of an unsupervised classification approach for the purpose of automated microarray data quality assessment. Since our approach requires only unannotated training data, it is easy to customize and to keep up-to-date as technology evolves. In contrast to other "black box" classification systems, this method also allows for intuitive explanations.</p

Liquid biopsies come of age: towards implementation of circulating tumour DNA

Improvements in genomic and molecular methods are expanding the range of potential applications for circulating tumour DNA (ctDNA), both in a research setting and as a ‘liquid biopsy’ for cancer management. Proof-of-principle studies have demonstrated the translational potential of ctDNA for prognostication, molecular profiling and monitoring. The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA. This is an opportune time to appraise potential approaches to ctDNA analysis, and to consider their applications in personalized oncology and in cancer research.We would like to acknowledge the support of The University of Cambridge, Cancer Research UK (grant numbers A11906, A20240, A15601) (to N.R., J.D.B.), the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. 337905 (to N.R.), the Cambridge Experimental Cancer Medicine Centre, and Hutchison Whampoa Limited (to N.R.), AstraZeneca (to R.B., S.P.), the Cambridge Experimental Cancer Medicine Centre (ECMC) (to R.B., S.P.), and NIHR Biomedical Research Centre (BRC) (to R.B., S.P.). J.G.C. acknowledges clinical fellowship support from SEOM