Optimising the analysis of transcript data using high density oligonucleotide arrays and genomic DNA-based probe selection

Background: Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe-set, consisting of up to 16 probe-pairs. Signal intensities across probe-pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results: Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion: This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays

Pigeons: a novel GUI software for analysing and parsing high density heterologous oligonucleotide microarray probe level data

Genomic DNA-based probe selection by using high density oligonucleotide arrays has recently been applied to heterologous species (Xspecies). With the advent of this new approach, researchers are able to study the genome and transcriptome of a non-model or an underutilised crop species through current state-of-the-art microarray platforms. However, a software package with a graphical user interface (GUI) to analyse and parse the oligonucleotide probe pair level data is still lacking when an experiment is designed on the basis of this cross species approach. A novel computer program called Pigeons has been developed for customised array data analysis to allow the user to import and analyse Affymetrix GeneChip® probe level data through XSpecies. One can determine empirical boundaries for removing poor probes based on genomic hybridisation of the test species to the Xspecies array, followed by making a species-specific Chip Description File (CDF) file for transcriptomics in the heterologous species, or Pigeons can be used to examine an experimental design to identify potential Single-Feature Polymorphisms (SFPs) at the DNA or RNA level. Pigeons is also focused around visualization and interactive analysis of the datasets. The software with its manual (the current release number version 1.2.1) is freely available at the website of the Nottingham Arabidopsis Stock Centre (NASC)

Parameter estimation for robust HMM analysis of ChIP-chip data

Tiling arrays are an important tool for the study of transcriptional activity, protein-DNA interactions and chromatin structure on a genome-wide scale at high resolution. Although hidden Markov models have been used successfully to analyse tiling array data, parameter estimation for these models is typically ad hoc. Especially in the context of ChIP-chip experiments, no standard procedures exist to obtain parameter estimates from the data. Common methods for the calculation of maximum likelihood estimates such as the Baum-Welch algorithm or Viterbi training are rarely applied in the context of tiling array analysis. Results: Here we develop a hidden Markov model for the analysis of chromatin structure ChIP-chip tiling array data, using t emission distributions to increase robustness towards outliers. Maximum likelihood estimates are used for all model parameters. Two different approaches to parameter estimation are investigated and combined into an efficient procedure. Conclusion: We illustrate an efficient parameter estimation procedure that can be used for HMM based methods in general and leads to a clear increase in performance when compared to the use of ad hoc estimates. The resulting hidden Markov model outperforms established methods like TileMap in the context of histone modification studies.13 page(s

Microarray analysis of tamoxifen resistance in breast cancer.

PhDTamoxifen remains a widely used hormone therapy for pre and postmenopausal women with hormone receptor positive breast cancer in both adjuvant and metastatic disease settings. Resistance to this well tolerated and cost-effective drug limits its use. Only an improved understanding of the mechanisms of tamoxifen resistance will provide the basis for overcoming this phenomenon. Expression profiles from tamoxifen-resistant and sensitive MCF7 derived breast cancer cell lines were prepared, using Affymetrix HG_U133A cDNA microarrays. The data generated was analysed to identify novel pathways and genes associated with tamoxifen resistance or sensitivity. Selected genes, whose expression correlates with response to tamoxifen, were validated using RT-PCR in cell lines and following this, in situ hybridisation and immunohistochemistry on cell lines. Functional analyses of these genes were carried out: genes that were down-regulated in tamoxifen resistant MCF7 cells (HRASLS3, CTSD, CAXII) were selectively knocked down using RNA interference. Cell lines stably over-expressing genes upregulated in the tamoxifen resistant MCF7s (ATP1B1, SOCS2, NR4A2) were selected. These manipulated cells were subsequently tested for their response to anti-oestrogen treatment. Another major marker in breast cancer is the ERBB2 proto-oncogene; overexpressed in 20% of breast carcinomas, it is associated with poor prognosis and hormone resistance. The transcriptional deregulation of ERBB2 in breast cancer may in part be mediated by the transcription factors AP-2 and . Previous studies have shown that ERBB2 expression is repressed by oestrogen activated oestrogen receptor and that AP-2 binding sites within the ERBB2 promoter and the intronic enhancer are required for this oestrogenic repression. Using RNA interference, AP-2 and were successfully knocked down in breast cancer cell lines MCF7, T47D and ZR75-1. These have been used to investigate the effect of AP-2 loss on ERBB2 expression in hormonally manipulated cells

Development of methods for combinational approaches to cis-regulatory module interactions

The complexity and size of the higher animal genome and relative scarcity of DNA-binding factors with which to regulate it imply a complex and pleiotropic regulatory system. Cisregulatory modules (CRMs) are vitally important regulators of gene expression in higher animal cells, integrating external and internal information to determine an appropriate response in terms of gene expression by means of direct and indirect interactions with the transcriptional machinery. The interaction space available within systems of multiple CRMs, each containing several sites where one or more factors could be bound is huge. Current methods of investigation involve the removal of individual sites or factors and measuring the resulting effect on gene expression. The effects of investigations of this type may be masked by the functional redundancy present in some of these regulatory systems as a result of their evolutionary development. The investigation of CRM function is limited by a lack of technology to generate and analyse combinatorial mutation libraries of CRMs, where putative transcription factor binding sites are mutated in various combinations to achieve a holistic view of how the factors binding to those sites cooperate to bring about CRM function. The principle work of this thesis is the generation of such a library. This thesis presents the development of microstereolithography as a method for making microfluidic devices, both directly and indirectly. A microfluidic device was fabricated that was used to generate oligonucleotide mixtures necessary to synthesise combinatorial mutants of a CRM sequence from the muscle regulatory factor MyoD. In addition, this thesis presents the development of the optimisation algorithms and assembly processes necessary for successful sequence assembly. Furthermore, it was found that the CRM, in combination with other CRMs, is able to synergistically regulate gene expression in a position and orientation independent manner in three separate contexts. Finally, by testing a small portion of the available combinatorial mutant library it was shown that mutation of individual binding sites within of the CRM is not sufficient to show a significant change in the level of reporter gene expression

Highlights from the Third International Society for Computational Biology Student Council Symposium at the Fifteenth Annual International Conference on Intelligent Systems for Molecular Biology

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract In this meeting report we give an overview of the 3rd International Society for Computational Biology Student Council Symposium. Furthermore, we explain the role of the Student Council and the symposium series in the context of large, international conferences.Published versio

The development of an electrochemical sensor for detecting and measuring circulating tumour DNA in human fluids

The high rates of mortality amongst cancer patients highlights the need for advances in rapid detection and enhanced point of care (PoC) testing. A simple approach tailored towards PoC cancer detection and monitoring using label-free electrochemical biosensors is presented. Screen-Printed Carbon electrodes (SPCEs) have been extensively employed as an economical transducer substrate for electrochemical biosensing applications due to their simplicity, affordability and versatility. In this work, a simple, low-cost DNA biosensor is presented which after initial work with Tp53 was developed specifically to detect mutations in a key oncogene (KRAS). Sensor arrays of SPCEs and carbon-nanotube (CNT) modified SPCEs were used to perform multiplexed measurements of DNA hybridisation. Various amplification techniques for enriching the pool of mutated DNA strands were explored and optimised. Amine-modified ssDNA probes were immobilized by modifying SPCEs and CNT-SPCEs with diazonium and EDC/NHS groups. The sensor performance was characterized using cyclic voltammetry, differential pulse voltammetry, square wave voltammetry and electrochemical impedance spectroscopy all to different extents. The detection principle was evaluated by showing effective on-chip DNA hybridization techniques, discrimination using negative controls, and performing multiple repetitions to ascertain reliability of the system. The developed sensor displayed some sensitivity and selectivity to Tp53, KRAS pG12D, and KRAS pG13D DNA, all of which are important mutations in cancer progression. For the amplified samples, 0.027 ng/µl amplicons were detectable while for the non-amplified samples, 0.85 ng/µl cfDNA concentration was detectable using the assay developed. The importance of these findings lies in the design of future electrochemical assays that are capable of discriminating between circulating tumour DNA in the blood prior to and post cancer therapy. The real-world application of this concept provides not only early diagnostic capability but an avenue for treatment decisions to be guided in such a way that health care providers can initiate, choose, avoid, alter or cease selected therapies when caring for patients that have shown symptoms for cancer or who are at risk of having recurrent cancers.The high rates of mortality amongst cancer patients highlights the need for advances in rapid detection and enhanced point of care (PoC) testing. A simple approach tailored towards PoC cancer detection and monitoring using label-free electrochemical biosensors is presented. Screen-Printed Carbon electrodes (SPCEs) have been extensively employed as an economical transducer substrate for electrochemical biosensing applications due to their simplicity, affordability and versatility. In this work, a simple, low-cost DNA biosensor is presented which after initial work with Tp53 was developed specifically to detect mutations in a key oncogene (KRAS). Sensor arrays of SPCEs and carbon-nanotube (CNT) modified SPCEs were used to perform multiplexed measurements of DNA hybridisation. Various amplification techniques for enriching the pool of mutated DNA strands were explored and optimised. Amine-modified ssDNA probes were immobilized by modifying SPCEs and CNT-SPCEs with diazonium and EDC/NHS groups. The sensor performance was characterized using cyclic voltammetry, differential pulse voltammetry, square wave voltammetry and electrochemical impedance spectroscopy all to different extents. The detection principle was evaluated by showing effective on-chip DNA hybridization techniques, discrimination using negative controls, and performing multiple repetitions to ascertain reliability of the system. The developed sensor displayed some sensitivity and selectivity to Tp53, KRAS pG12D, and KRAS pG13D DNA, all of which are important mutations in cancer progression. For the amplified samples, 0.027 ng/µl amplicons were detectable while for the non-amplified samples, 0.85 ng/µl cfDNA concentration was detectable using the assay developed. The importance of these findings lies in the design of future electrochemical assays that are capable of discriminating between circulating tumour DNA in the blood prior to and post cancer therapy. The real-world application of this concept provides not only early diagnostic capability but an avenue for treatment decisions to be guided in such a way that health care providers can initiate, choose, avoid, alter or cease selected therapies when caring for patients that have shown symptoms for cancer or who are at risk of having recurrent cancers

Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide

BACKGROUND: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. METHODS: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. RESULTS: Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. CONCLUSIONS: Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. GENERAL SIGNIFICANCE: Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani