Toward learning the principles of plant gene regulation

Advanced machine learning (ML) algorithms produce highly accurate models of gene expression, uncovering novel regulatory features in nucleotide sequences involving multiple cis-regulatory regions across whole genes and structural properties. These broaden our understanding of gene regulation and point to new principles to test and adopt in the field of plant science

Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms

<p>Abstract</p> <p>Background</p> <p>The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan^®and SYBR^®Green real-time PCR chemistries. In our study four alternative chemistries: Lux™, Plexor™, Cycling Probe Technology and LNA^®were extensively evaluated and compared using TaqMan^®chemistry as a reference system.</p> <p>Results</p> <p>Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics.</p> <p>Conclusion</p> <p>Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA^®technology is an alternative to TaqMan^®when designing assays for quantitative analysis. Because LNA^®probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan^®probe is difficult or even impossible due to sequence characteristics. Plexor™ on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.</p

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

BACKGROUND: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. RESULTS: Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. CONCLUSION: The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification

Discovery of relevant response in infected potato plants from time series of gene expression data

The paper presents a methodology for analyzing time series of gene expression data collected from the leaves of potato virus Y (PVY) infected and non-infected potato plants, with the aim to identify significant differences between the two sets of potato plants’ characteristic for various time points. We aim at identifying differentially- expressed genes whose expression values are statistically significantly different in the set of PVY infected potato plants compared to non- infected plants, and which demonstrate also statistically significant changes of expression values of genes of PVY infected potato plants in time. The novelty of the approach includes stratified data randomization used in estimating the statistical properties of gene expression of the samples in the control set of non-infected potato plants. A novel estimate that computes the relative minimal distance between the samples has been defined that enables reliable identification of the differences between the target and control datasets when these sets are small. The relevance of the outcomes is demonstrated by visualizing the relative minimal distance of gene expression changes in time for three different types of potato leaves for the genes that have been identified as relevant by the proposed methodology

Adaptation of the MapMan ontology to biotic stress responses: application in solanaceous species

<p>Abstract</p> <p>Background</p> <p>The results of transcriptome microarray analysis are usually presented as a list of differentially expressed genes. As these lists can be long, it is hard to interpret the desired experimental treatment effect on the physiology of analysed tissue, e.g. via selected metabolic or other pathways. For some organisms, gene ontologies and data visualization software have been implemented to overcome this problem, whereas for others, software adaptation is yet to be done.</p> <p>Results</p> <p>We present the classification of tentative potato contigs from the potato gene index (StGI) available from Dana-Farber Cancer Institute (DFCI) into the MapMan ontology to enable the application of the MapMan family of tools to potato microarrays. Special attention has been focused on mapping genes that could not be annotated based on similarity to Arabidopsis genes alone, thus possibly representing genes unique for potato. 97 such genes were classified into functional BINs (i.e. functional classes) after manual annotation. A new pathway, focusing on biotic stress responses, has been added and can be used for all other organisms for which mappings have been done. The BIN representation on the potato 10 k cDNA microarray, in comparison with all putative potato gene sequences, has been tested. The functionality of the prepared potato mapping was validated with experimental data on plant response to viral infection. In total 43,408 unigenes were mapped into 35 corresponding BINs.</p> <p>Conclusion</p> <p>The potato mappings can be used to visualize up-to-date, publicly available, expressed sequence tags (ESTs) and other sequences from GenBank, in combination with metabolic pathways. Further expert work on potato annotations will be needed with the ongoing EST and genome sequencing of potato. The current MapMan application for potato is directly applicable for analysis of data obtained on potato 10 k cDNA microarray by TIGR (The Institute for Genomic Research) but can also be used by researchers working on other potato gene sets. The potato mapping file and the stress mapping diagram are available from the MapMan website <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>.</p

Interactive exploration of heterogeneous biological networks with Biomine Explorer

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Bisociative knowledge discovery for microarray data analysis

Peer reviewe

OPHTHALMOSCOPIC EVALUATION OF THE OPTIC NERVE HEAD

Background. Optic nerve diseases, such as glaucomas, lead to changes in the intrapapillary and parapapillary region of the optic nerve head.Conclusions. These changes can be described by the following variables: size and shape of the optic disc; size, shape, and pallor of the neuroretinal rim; size of the optic cup in relation to the area of the disc; configuration and depth of the optic cup; ratios of cup to disc diameter and cup to disc area; position of the exit of the central retinal vessel trunk on the lamina cribrosa surface; presence and location of splinter-shaped hemorrhages; occurence, size, configuration, and location of parapapillary chorioretinal atrophy; diffuse and/or focal decrease of the diameter of the retinal arterioles; and visibility of the retinal nerve fiber layer (RNFL). These variables can be assessed semiquantitively by ophthalmoscopy without applying sophisticated techniques. For the early detection of glaucomatous optic nerve damage in ocular hypertensive eyes before the development of visual field loss, the most important variables are neuroretinal rim shape, optic cup size in relation to optic disc size, diffusely or segmentally decreased visibility of the RNFL, occurence of localized RNFL defects, and presence of disc hemorrhages.</p

Improvement of root architecture under abiotic stress through control of auxin homeostasis in Arabidopsis and Brassica crops

Auxin plays an important role in many aspects of plant development including stress responses. Here we briefly summarize how auxin is involved in salt stress, drought (i.e. mostly osmotic stress), waterlogging and nutrient deficiency in Brassica plants. In addition, some mechanisms to control auxin levels and signaling in relation to root formation (under stress) will be reviewed. Molecular studies are mainly described for the model plant Arabidopsis thaliana, but we also like to demonstrate how this knowledge can be transferred to agriculturally important Brassica species, such as Brassica rapa, Brassica napus and Brassica campestris. Moreover, beneficial fungi could play a role in the adaptation response of Brassica roots to abiotic stresses. Therefore, the possible influence of Piriformospora indica will also be covered since the growth promoting response of plants colonized by P. indica is also linked to plant hormones, among them auxin