assessment-of-dna-contamination-in-rna-samples-based-on-ribosomal-dna

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

obtaining-high-quality-transcriptome-data-from-cereal-seeds-modified

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)

In plants, RNA-directed DNA methylation (RdDM) and related transcriptional gene silencing (TGS) involve members of the suppressor of variegation 3-9-homologous (SUVH) group of putative histone methyltransferases. Utilizing a reverse genetic approach in Arabidopsis thaliana, we demonstrate that two closely related SUVH members, SUVH2 and SUVH9, act partially non-redundant in RdDM. DNA methylation, transcript accumulation and association with histone modifications were analyzed at the endogenous RdDM target AtSN1 (a SINE-like retroelement) in suvh2 and suvh9 single as well as suvh2 suvh9 double mutants. SUVH2 was found to be required for full DNA methylation at AtSN1 in early seed development and was also higher expressed in seeds than at later developmental stages. SUVH9 had its impact on RdDM later during vegetative development of the plant and was also higher expressed during that stage than at earlier developmental stages. The strongest reduction of RdDM at AtSN1 was found in suvh2 suvh9 double mutant plants. Histone 3-lysine 9-dimethylation (H3K9me2) associated with AtSN1 was reduced only in the simultaneous absence of functional SUVH2 and SUVH9. Thus, SUVH2 and SUVH9 functions in RdDM and TGS are overlapping in spite of some developmental specialization. Pol V specific transcripts were reduced in suvh2 suvh9 plants. This might indicate a role of these SUVH proteins in Pol V complex recruitment.

Developmentally non-redundant SET domain proteins SUVH2 and SUVH9 are required for transcriptional gene silencing in Arabidopsis thaliana.

DNA methylation is an important biomarker for gene activity. It contributes to gene silencing and is involved in regulating various seed developmental processes in plants. Many of these processes are involved in important traits associated with aspects of grain quality. A reliable, fast, and cheap method is the estimation of DNA methylation utilizing methylation sensitive restriction enzymes (MSRE) and quantitative real-time PCR (qPCR) for selected candidate regions. The presented method can be used to confirm an effect of RNAi constructs on their target genes or trans-activity. Analysis of promoter regions can contribute to estimation of gene activity and related traits.

Markus Kuhlmann

RG Abiotic Stress Genomics/ RG Heterosis,
Department Molecular Genetics,
Department of Molecular Genetics,
Heterosis Research Group,
RG Abiotic Stress Genomics/ RG Heterosis, Department Molecular Genetics,
Department of Molecular Genetics, Heterosis Research Group

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling