Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data

<p>Abstract</p> <p>Background</p> <p>Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RT-qPCR) is the most accurate measure of gene expression in biological systems. The comparison of different samples requires the transformation of data through a process called normalisation. Reference or housekeeping genes are candidate genes which are selected on the basis of constitutive expression across samples, and allow the quantification of changes in gene expression. At present, no reference gene has been identified for any organism which is universally optimal for use across different tissue types or disease situations. We used microarray data to identify new reference genes generated from total RNA isolated from normal and osteoarthritic canine articular tissues (bone, ligament, cartilage, synovium and fat). RT-qPCR assays were designed and applied to each different articular tissue. Reference gene expression stability and ranking was compared using three different mathematical algorithms.</p> <p>Results</p> <p>Twelve new potential reference genes were identified from microarray data. One gene (mitochondrial ribosomal protein S7 [<it>MRPS7</it>]) was stably expressed in all five of the articular tissues evaluated. One gene HIRA interacting protein 5 isoform 2 [<it>HIRP5</it>]) was stably expressed in four of the tissues evaluated. A commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>) was not stably expressed in any of the tissues evaluated. Most consistent agreement between rank ordering of reference genes was observed between <it>Bestkeeper©</it> and geNorm, although each method tended to agree on the identity of the most stably expressed genes and the least stably expressed genes for each tissue. New reference genes identified using microarray data normalised in a conventional manner were more stable than those identified by microarray data normalised by using a real-time RT-qPCR methodology.</p> <p>Conclusion</p> <p>Microarray data normalised by a conventional manner can be filtered using a simple stepwise procedure to identify new reference genes, some of which will demonstrate good measures of stability. Mitochondrial ribosomal protein S7 is a new reference gene worthy of investigation in other canine tissues and diseases. Different methods of reference gene stability assessment will generally agree on the most and least stably expressed genes, when co-regulation is not present.</p

Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer

Introduction: The purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets. Methods: Analysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription. Results: TXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species. Conclusions: TXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner

Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data-0

<p><b>Copyright information:</b></p><p>Taken from "Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data"</p><p>http://www.biomedcentral.com/1471-2199/8/62</p><p>BMC Molecular Biology 2007;8():62-62.</p><p>Published online 25 Jul 2007</p><p>PMCID:PMC1976117.</p><p></p> changes in expression between experimental groups, the co-efficient of variation and ontological evaluatio