Representational Difference AnalysisA Methodology to Study Differential Gene Expression

Frohme, Marcus; Hoheisel, Joörg D.

doi:10.1016/b978-012164730-8/50200-8

Cited by 3 publications

(3 citation statements)

References 26 publications

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“…RDA as a rapid PCR-based method [19] has been successfully adapted and used for the cloning of genes, which are differentially expressed between active and inactive animals. This method is particularly appropriate for application to species whose genomes and expressed sequences have been very little characterized [24].…”

Section: Resultsmentioning

confidence: 99%

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Genomic Analyses of Cryptobiotic Tardigrades

Mali¹,

Grohme²,

Wełnicz³

et al. 2008

Wissenschaftliche Beiträge / Technische Hochschule Wildau

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Techniken der Genomanalyse, speziell das transkriptionelle Profi ling, erlauben uns, Genexpression innerhalb eines und zwischen verschiedenen Organismen zu untersuchen. Wir nutzen als Modell Tardigraden, auch bekannt als Bärtierchen, um das Phänomen der Kryptobiose besser zu verstehen. Die ausgetrockneten Tiere können Jahre überdauern. Wenn man Wasser hinzugibt, rehydrieren die Tiere und werden wieder zum Leben erweckt. In unserem Labor untersuchen wir die Genexpression als Antwort auf das Austrockenen und Rehydrieren mittels Bibliotheken Exprimierter Sequenz Tags (ESTs), Repräsentativer Differenz Analyse und Mikroarray-Untersuchungen. Die molekulare Analyse des komplexen Phänomens und seiner Regulation auf Ebene der Gene wird es ermöglichen, Techniken für die Konservierung und Stabilisierung von biologischem Material in trockenem Zustand zu entwickeln. Summary Genomics technologies, especially transcriptional profi ling, allows comparisons of gene expression within and across different organisms. By the use of the model group Tardigrada, also known as water bear, we try to understand the phenomenon of cryptobiosis. The dry organisms can survive for years without water. When re-exposed to water, the animals rehydrate and come back to life. The expression of genes in response to dehydratation and rehydratation is being examined in our laboratory through the generation of expressed sequence tags (ESTs), representational difference analysis (RDA) and subsequent microarray analysis. Molecular dissection of this complex phenomenom, including gene regulation, will allow the development of techniques for preservation and stabilisation of biological materials in a dried state.

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Section: Resultsmentioning

confidence: 99%

“…We used representational difference analysis (RDA), which couples substractive hybridization to PCR-mediated kinetic enrichment [19], to identify differentially expressed genes between active and inactive tardigrades. cDNA for RDA was prepared from both states.…”

Section: Methodsmentioning

confidence: 99%

Genomic Analyses of Cryptobiotic Tardigrades

Mali¹,

Grohme²,

Wełnicz³

et al. 2008

Wissenschaftliche Beiträge / Technische Hochschule Wildau

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“…cDNA RDA was performed as previously published (Frohme and Hoheisel 2006; a detailed protocol is available from the authors) using Dpn II for generating representations from 1 μg of SMART TM amplified cDNA. The subtraction and enrichment steps were performed in three consecutive rounds with tester : driver ratios of 1 : 100, 1 : 1000 and 1 : 400 000.…”

Section: Methodsmentioning

confidence: 99%

cDNA representational difference analysis for identifying transcripts regulated under anhydrobiosis in the tardigrade Milnesium tardigradum

Grohme

Mali

Schill

et al. 2011

Journal of Zoological Systematics and Evolutionary Research

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Although water is a prerequisite for life, some organisms are capable of surviving complete cellular dehydration by entering a state called anhydrobiosis. In this state, no metabolic activity is measurable and these organisms can survive several years in dry form until water becomes available again. Anhydrobiosis has been described in bacteria, fungi, plants and many invertebrates. Among those invertebrates, tardigrades are also known to be capable to undergo anhydrobiosis by forming a so-called ÔtunÕ stage. Therefore, they represent an interesting model system for studying the cellular response to desiccation. We have applied cDNA representational difference analysis to investigate the transcriptional response of the tardigrade Milnesium tardigradum to desiccation. This resulted in the identification of two transcripts that showed significant regulation between active and anhydrobiotic animals as validated by quantitative real-time PCR. One of these putative transcripts was downregulated fivefold. It did not show any significant similarity to known transcripts, but had previously been cloned in M. tardigradum. The second transcript could be identified as the M. tardigradum homologue of a subunit of replication factor C and was downregulated more than sixfold in the anhydrobiotic state. Replication factor C plays an important role in DNA replication during cell division. Both candidates showed an even lower expression in the transition stage between active and the tun stage (27-and 25-fold reduction, respectively), implying active inhibition of transcription of these two transcripts. Our results suggest that the potential modulation of DNA replication and its role in protecting genome stability should attract more attention when studying anhydrobiotic animals.

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Methylation levels of a novel genetic element, EgNB3 as a candidate biomarker associated with the embryogenic competency of oil palm

Ooi

Mayes

et al. 2013

Tree Genetics & Genomes

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The association between DNA methylation status and embryogenic competency in oil palm tissue culture was examined through Representational Difference Analysis (RDA) approach, using methylation-sensitive restriction endonucleases. "Difference Products" (DPs) of RDA derived from palms of similar genetic backgrounds but exhibiting different embryogenesis rates during the regeneration process were isolated. The DPs were sequenced using a pyrosequencing platform. To our knowledge, this is the first study profiling partial HpaII methylation sites in oil palm young leaf tissues which are potentially associated with embryogenic amenability through a genomic subtractive approach. Quantitative real-time PCR analysis demonstrated that the methylation status of a novel fragment, EgNB3, was higher in highly embryogenic leaf explants compared to low embryogenesis rate materials. These differences are likely to be contributed by the 5′-mCCGG-3′ and/or 5′-mCmCGG-3′ methylation patterns. Our data suggest that the differentially methylated site in EgNB3 has potential as a molecular biomarker for the screening of oil palm leaf explants for their embryogenic potentials.

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Representational Difference AnalysisA Methodology to Study Differential Gene Expression

Cited by 3 publications

References 26 publications

Genomic Analyses of Cryptobiotic Tardigrades

Genomic Analyses of Cryptobiotic Tardigrades

cDNA representational difference analysis for identifying transcripts regulated under anhydrobiosis in the tardigrade Milnesium tardigradum

Methylation levels of a novel genetic element, EgNB3 as a candidate biomarker associated with the embryogenic competency of oil palm

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