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Coll A.,University of Girona | Collado R.,University of Girona | Capellades G.,Fundacio Mas Badia | Kubista M.,Academy of Sciences of the Czech Republic | And 2 more authors.
Plant Molecular Biology | Year: 2010

The introduction of genetically modified organisms (GMO) in many countries follows strict regulations to ensure that only safety-tested products are marketed. Over the last few years, targeted approaches have been complemented by profiling methods to assess possible unintended effects of transformation. Here we used a commercial (Affymertix) microarray platform (i.e. allowing assessing the expression of ~1/3 of the genes of maize) to evaluate transcriptional differences between commercial MON810 GM maize and non-transgenic crops in real agricultural conditions, in a region where about 70% of the maize grown was MON810. To consider natural variation in gene expression in relation to biotech plants we took two common MON810/non-GM variety pairs as examples, and two farming practices (conventional and low-nitrogen fertilization). MON810 and comparable non-GM varieties grown in the field have very low numbers of sequences with differential expression, and their identity differs among varieties. Furthermore, we show that the differences between a given MON810 variety and the non-GM counterpart do not appear to depend to any major extent on the assayed cultural conditions, even though these differences may slightly vary between the conditions. In our study, natural variation explained most of the variability in gene expression among the samples. Up to 37.4% was dependent upon the variety (obtained by conventional breeding) and 31.9% a result of the fertilization treatment. In contrast, the MON810 GM character had a very minor effect (9.7%) on gene expression in the analyzed varieties and conditions, even though similar cryIA(b) expression levels were detected in the two MON810 varieties and nitrogen treatments. This indicates that transcriptional differences of conventionally-bred varieties and under different environmental conditions should be taken into account in safety assessment studies of GM plants. © 2010 Springer Science+Business Media B.V.

Pazzagli M.,University of Florence | Malentacchi F.,University of Florence | Simi L.,University of Florence | Orlando C.,University of Florence | And 11 more authors.
Methods | Year: 2013

The diagnostic use of in vitro molecular assays can be limited by the lack of guidelines for collection, handling, stabilization and storage of patient specimens. One of the major goals of the EC funded project SPIDIA (www.spidia.eu) is to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for molecular testing which requires intracellular RNA analytes. To this end, a survey and a pan-European external quality assessment (EQA) were implemented. This report is the summary of the results of that trial. With the European Federation of Laboratory Medicine (EFLM) support, 124 applications for participation in the trial were received from 27 different European countries, and 102 laboratories actually participated in the trial. Each participating laboratory described their respective laboratory policies and practices as well as blood collection tubes typically used in performing this type of testing. The participating laboratories received two identical blood specimens: in an EDTA tubes (unstabilized blood; n= 67) or in tubes designed specifically for the stabilization of intracellular RNA in blood (PAXgene® Blood RNA tubes; n= 35). Laboratories were requested to perform RNA extraction according to the laboratory's own procedure as soon as possible upon receipt of the tubes for one tube and 24. h after the first extraction for the second tube. Participants (n= 93) returned the two extracted RNAs to SPIDIA facility for analysis, and provided details about the reagents and protocols they used for the extraction. At the SPIDIA facility responsible for coordinating the study, the survey data were classified, and the extracted RNA samples were evaluated for purity, yield, integrity, stability, and the presence of interfering substances affecting RT-qPCR assays. All participants received a report comparing the performance of the RNA they submitted to that of the other participants. All the results obtained by participants for each RNA quality parameter were classified as "in control", "warning", "out of control" and "missing" by consensus mean analysis. From the survey data, the most variable parameters were the volume of blood collected and the time and storage temperature between blood collection and RNA extraction. Analyzing the results of quality testing of submitted RNA samples we observed a data distribution of purity, yield, and presence of assay interference in agreement with expected values. The RNA Integrity Number (RIN) values distribution was, on the other hand, much wider than the optimal expected value, which led to an "in control" classification, even for partly degraded RNA samples. On the other hand, RIN values below 5 significantly correlated with a reduction of GAPDH expression levels. Furthermore, the distribution of the values of the four transcripts investigated (c-fos, IL-1β, IL-8, and GAPDH) was wide and the RNA instability between samples separated by 24. h were similar. Assuming the presence of at least two quality parameters "out of control" as an indication of a critical performance of the laboratory, 33% of the laboratories were included in this group. The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the pre-analytical phase of RNA analysis of blood samples. © 2012 Elsevier Inc.

Virant-Klun I.,University of Ljubljana | Skutella T.,University of Heidelberg | Kubista M.,Biocenter | Kubista M.,Academy of Sciences of the Czech Republic | And 3 more authors.
BioMed Research International | Year: 2013

The aim of this study was to trigger the expression of genes related to oocytes in putative ovarian stem cells scraped from the ovarian surface epithelium of women with premature ovarian failure and cultured in vitro in the presence of follicular fluid, rich in substances for oocyte growth and maturation. Ovarian surface epithelium was scraped and cell cultures were set up by scrapings in five women with nonfunctional ovaries and with no naturally present mature follicles or oocytes. In the presence of donated follicular fluid putative stem cells grew and developed into primitive oocyte-like cells. A detailed single-cell gene expression profiling was performed to elucidate their genetic status in comparison to human embryonic stem cells, oocytes, and somatic fibroblasts. The ovarian cell cultures depleted/converted reproductive hormones from the culture medium. Estradiol alone or together with other substances may be involved in development of these primitive oocyte-like cells. The majority of primitive oocyte-like cells was mononuclear and expressed several genes related to pluripotency and oocytes, including genes related to meiosis, although they did not express some important oocyte-specific genes. Our work reveals the presence of putative stem cells in the ovarian surface epithelium of women with premature ovarian failure. © 2013 Irma Virant-Klun et al.

Flachsova M.,Academy of Sciences of the Czech Republic | Flachsova M.,Charles University | Sindelka R.,Academy of Sciences of the Czech Republic | Kubista M.,Academy of Sciences of the Czech Republic | Kubista M.,Biocenter
Scientific Reports | Year: 2013

We have measured the expression of 41 maternal mRNAs in individual blastomeres collected from the 8 to 32-cellXenopus laevisembryos to determine when and how asymmetry in the body plan is introduced. We demonstrate that the asymmetry along the animal-vegetal axis in the oocyte is transferred to the daughter cells during early cell divisions. All studied mRNAs are distributed evenly among the set of animal as well as vegetal blastomeres. We find no asymmetry in mRNA levels that might be ascribed to the dorso-ventral specification or the left-right axis formation. We hypothesize that while the animal-vegetal asymmetry is a consequence of mRNA gradients, the dorso-ventral and left-right axes specifications are induced by asymmetric distribution of other biomolecules, probably proteins.

Sindelka R.,Whitehead Institute For Biomedical Research | Sidova M.,Academy of Sciences of the Czech Republic | Sidova M.,Charles University | Svec D.,Academy of Sciences of the Czech Republic | And 2 more authors.
Methods | Year: 2010

qPCR tomography was developed to study mRNA localization in complex biological samples that are embedded and cryo-sectioned. After total RNA extraction and reverse transcription, the spatial profiles of mRNAs and other functional RNAs were determined by qPCR. The Xenopus laevis oocyte was selected as model, because of its large size (more than 1 mm) and large amount of total RNA (∼5 μg). Fifteen sections along the animal-vegetal axis were cut and prepared for quantification of 31 RNA targets using the high-throughput real-time RT-PCR (qPCR) BioMark™ platform. mRNAs were found to have two localization patterns, animal/central or vegetal. Because of the high resolution in sectioning, it was possible to distinguish two subgroups of the vegetal gene patterns: germ plasm determinant pattern and profile of other vegetal genes. © 2010 Elsevier Inc. All rights reserved.

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