Error, reproducibility and sensitivity : a pipeline for data processing of Agilent oligonucleotide expression arrays

Background Expression microarrays are increasingly used to obtain large scale transcriptomic information on a wide range of biological samples. Nevertheless, there is still much debate on the best ways to process data, to design experiments and analyse the output. Furthermore, many of the more sophisticated mathematical approaches to data analysis in the literature remain inaccessible to much of the biological research community. In this study we examine ways of extracting and analysing a large data set obtained using the Agilent long oligonucleotide transcriptomics platform, applied to a set of human macrophage and dendritic cell samples. Results We describe and validate a series of data extraction, transformation and normalisation steps which are implemented via a new R function. Analysis of replicate normalised reference data demonstrate that intrarray variability is small (only around 2% of the mean log signal), while interarray variability from replicate array measurements has a standard deviation (SD) of around 0.5 log2 units ( 6% of mean). The common practise of working with ratios of Cy5/Cy3 signal offers little further improvement in terms of reducing error. Comparison to expression data obtained using Arabidopsis samples demonstrates that the large number of genes in each sample showing a low level of transcription reflect the real complexity of the cellular transcriptome. Multidimensional scaling is used to show that the processed data identifies an underlying structure which reflect some of the key biological variables which define the data set. This structure is robust, allowing reliable comparison of samples collected over a number of years and collected by a variety of operators. Conclusions This study outlines a robust and easily implemented pipeline for extracting, transforming normalising and visualising transcriptomic array data from Agilent expression platform. The analysis is used to obtain quantitative estimates of the SD arising from experimental (non biological) intra- and interarray variability, and for a lower threshold for determining whether an individual gene is expressed. The study provides a reliable basis for further more extensive studies of the systems biology of eukaryotic cells

Identification of the Receptor Used by the Ecotropic Mouse GLN Endogenous Retrovirus

International audienceApproximately 10% of the mouse genome is composed of endogenousretroviruses belonging to different families. In contrast to the situation in the humangenome, several of these families correspond to recent, still-infectious elements capable of encoding complete viral particles. The mouse GLN endogenous retrovirus isone of these active families. We previously identified one fully functional provirusfrom the sequenced genome of the C57BL/6 mouse strain. The GLN envelope protein gives the infectious viral particles an ecotropic host range, and we had demonstrated that the receptor was neither CAT1 nor SMIT1, the two previously identifiedreceptors for mouse ecotropic retroviral envelope proteins. In this study, we haveidentified SLC19A1, the reduced folate carrier, as the cellular protein used as a receptor by the GLN retrovirus. The ecotropic tropism exhibited by this envelope isdue to the presence or absence of an N-linked glycosylation site in the first extracellular loop as well as the specific amino acid sequence of the extracellular domains ofthe receptor. Like all the other retroviral envelope proteins from the gammaretrovirus genus whose receptors have been identified, the GLN envelope protein uses amember of the solute carrier superfamily as a receptor

Discrimination of Geographical Origin of Asian Garlic Using Isotopic and Chemical Datasets under Stepwise Principal Component Analysis

Isotopic compositions of H-2, O-18, C-13, and N-15 and concentrations of 22 trace elements from garlic samples were analyzed and processed with stepwise principal component analysis (PCA) to discriminate garlic's country of origin among Asian regions including South Korea, Vietnam, Taiwan, and China. Results indicate that there is no single trace-element concentration or isotopic composition that can accomplish the study's purpose and the stepwise PCA approach proposed does allow for discrimination between countries on a regional basis. Sequentially, Step-1 PCA distinguishes garlic's country of origin among Taiwanese, South Korean, and Vietnamese samples; Step-2 PCA discriminates Chinese garlic from South Korean garlic; and Step-3 and Step-4 PCA, Chinese garlic from Vietnamese garlic. In model tests, countries of origin of all audit samples were correctly discriminated by stepwise PCA. Consequently, this study demonstrates that stepwise PCA as applied is a simple and effective approach to discriminating country of origin among Asian garlics

Discrimination of Geographical Origin of Asian Garlic Using Isotopic and Chemical Datasets under Stepwise Principal Component Analysis

Isotopic compositions of δ2 H, δ18 O, δ13 C, and δ15 N and concentrations of 22 trace elements from garlic samples were analyzed and processed with stepwise principal component analysis (PCA) to discriminate garlic's country of origin among Asian regions including South Korea, Vietnam, Taiwan, and China. Results indicate that there is no single trace-element concentration or isotopic composition that can accomplish the study's purpose and the stepwise PCA approach proposed does allow for discrimination between countries on a regional basis. Sequentially, Step-1 PCA distinguishes garlic's country of origin among Taiwanese, South Korean, and Vietnamese samples; Step-2 PCA discriminates Chinese garlic from South Korean garlic; and Step-3 and Step-4 PCA, Chinese garlic from Vietnamese garlic. In model tests, countries of origin of all audit samples were correctly discriminated by stepwise PCA. Consequently, this study demonstrates that stepwise PCA as applied is a simple and effective approach to discriminating country of origin among Asian garlics

A human endogenous retrovirus-derived gene that can contribute to oncogenesis by activating the ERK pathway and inducing migration and invasion

<div><p>Endogenous retroviruses are cellular genes of retroviral origin captured by their host during the course of evolution and represent around 8% of the human genome. Although most are defective and transcriptionally silenced, some are still able to generate retroviral-like particles and proteins. Among these, the HERV-K(HML2) family is remarkable since its members have amplified relatively recently and many of them still have full length coding genes. Furthermore, they are induced in cancers, especially in melanoma, breast cancer and germ cell tumours, where viral particles, as well as the envelope protein (Env), can be detected. Here we show that HERV-K(HML2) Env <i>per se</i> has oncogenic properties. Its expression in a non-tumourigenic human breast epithelial cell line induces epithelial to mesenchymal transition (EMT), often associated with tumour aggressiveness and metastasis. In our model, this is typified by key modifications in a set of molecular markers, changes in cell morphology and enhanced cell motility. Remarkably, microarrays performed in 293T cells reveal that HERV-K(HML2) Env is a strong inducer of several transcription factors, namely ETV4, ETV5 and EGR1, which are downstream effectors of the MAPK ERK1/2 and are associated with cellular transformation. We demonstrate that HERV-K(HML2) Env effectively activates the ERK1/2 pathway in our experimental setting and that this activation depends on the Env cytoplasmic tail. In addition, this phenomenon is very specific, being absent with every other retroviral Env tested, except for Jaagsiekte Sheep Retrovirus (JSRV) Env, which is already known to have transforming properties <i>in vivo</i>. Though HERV-K Env is not directly transforming by itself, the newly discovered properties of this protein may contribute to oncogenesis.</p></div

Oncogenic properties carried by different retroviral Envs.

<p><b>(A)</b> Phosphorylation of ERK1/2 in 293T cells expressing a panel of retroviral Envs was assayed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006451#ppat.1006451.g003" target="_blank">Fig 3B</a>. Samples in left and right panels were prepared and processed (migration, transfer, revelation) at the same time; therefore intensity of the signals in both sets of panels can be compared directly. The Envs tested span three different retroviral genera and are colour-coded according to group (red: betaretroviruses; blue: gammaretrovirus; purple: deltaretrovirus). <b>(B)</b> The mRNA levels of the transcription factors EGR1, ETV4 and ETV5 following expression of the indicated Envs were measured as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006451#ppat.1006451.g002" target="_blank">Fig 2C</a>. Error bars represent standard deviation of the mean of three independent experiments. (<b>C</b>) The transforming activity of Ampho, HERV-K, JSRV and enJSRV retroviral Env proteins was assessed in 208F cells. Briefly, cells were transiently transfected with expression vectors for the 4 Env proteins and left to reach confluence. The number of transformed foci was counted after 3–4 weeks. The histogram represents the average number of foci obtained per 2x10⁵ cells in 3 independent experiments. (<b>D</b>) Phosphorylation of ERK1/2 in 293T cells expressing the 4 retroviral Envs mentioned above was assessed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006451#ppat.1006451.g003" target="_blank">Fig 3B</a>. (<b>E</b>) The mRNA levels of transfection factors EGR1, ETV4 and ETV5 following expression of the 4 Envs was tested as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006451#ppat.1006451.g002" target="_blank">Fig 2C</a>. (<b>F</b>) We ensured that all the expression vectors for the Env proteins were functional by measuring the viral titres obtained with them when pseudotyping heterologous retroviral particles. Ampho, RD114, GaLV, FelV and MMTV Envs were tested using a MLV core, HERV-K, JSRV, enJSRV, IAP and HTLV1 Envs were tested with a HIV core. Viral titres were measured on 293T cells, except for FeLV pseudotypes (feline G355.5 cells) and MMTV pseudotypes (mouse NIH 3T3 cells). Background infection levels (i.e. the titres obtained with no Env expression vector) were less than 100 whatever the core/target cells combination.</p

Test for ERK1/2 phosphorylation and expression of transcription factors induced by endogenous HERV-K Env alleles.

<p><b>(A)</b> Previously described endogenous alleles of HERV-K Env were tested for their ability to induce phosphorylation of ERK1/2 by Western blot on 293T cell lysates following transient transfection. Expression of each HERV-K Env allele was also assessed. We also checked that Ampho Env was properly expressed. <b>(B)</b> Expression levels of the transcription factors EGR1, ETV4 and ETV5 induced by the endogenous alleles of HERV-K Env were measured as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006451#ppat.1006451.g002" target="_blank">Fig 2C</a>. Error bars represent the standard deviation of the mean of four independent experiments. (<b>C</b>) Recapitulation of the functional properties of all HERV-K Env-Rec variants which have been assessed [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006451#ppat.1006451.ref043" target="_blank">43</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006451#ppat.1006451.ref061" target="_blank">61</a>] (nd: not determined).</p

HERV-K envelope protein induces EMT in MCF10A human breast epithelial cell line.

<p>MCF10A cells were transduced with either a control “empty” vector (EV) or a vector expressing HERV-K Env-Rec. (<b>A</b>) Fluorescence microscopy images of MCF10A cell populations after selection with hygromycin compared to control wildtype cells (WT) and cells stimulated with 5ng/ml TGFβ (TGFβ). Images were taken at 10x magnification after staining the cells with Cell Tracker Green. (<b>B</b>) Changes in cell morphology were quantified by calculating the average cell size (area) of each population (a minimum of 1000 cells were counted for each population). (<b>C</b>) The expression levels of EMT related transcripts were evaluated in the different MCF10A populations by qRT-PCR. Genes of interest were normalized to the RPLO gene and expressed relative to WT cells. (<b>D</b>+<b>E</b>) The migration and invasion capacities of the cells were assessed using transwell assays. The cells were allowed to migrate and invade (through a layer of Matrigel) for 22 hours. DAPI staining was performed and cells that had migrated/invaded were observed under a fluorescent microscope and counted. Migration and invasion of TGF stimulated cells and HERV-K Env expressing cells is represented relative to WT and EV cells respectively. Error bars represent the standard deviation of at least 3 independently generated cell populations with triplicate readings taken from each population. Significant differences between cell populations were assessed using a one-tailed t test performed relative to WT cells (C+E) and EV cells (D); * = p<0.05; ** = p<0.01; *** = p<0.001.</p