Project description:Recent advances in (meta)genomic methods have provided new opportunities to examine host-microbe-environment interactions in the human gut. While opportunities exist to extract DNA from freshly sourced colonic tissue there are potentially valuable sources of DNA from historical studies that might also be examined. We examined how four different tissue DNA extraction methods employed in past clinical trials might impact the recovery of microbial DNA from a colonic tissue sample as assessed using a custom designed phylogenetic microarray for human gut bacteria and archaebacteria. While all methods of DNA extraction produced similar phylogenetic profiles some extraction specific biases were also observed. Real time PCR analysis targeting several bacterial groups substantiated this observation. These data suggest that while the efficacy of different DNA extraction methods differs somewhat all the methods tested produce an accurate representation of microbial diversity. This suggests that DNA samples archived in biobanks should be suitable for retrospective analyses.
Project description:We used microarray to investigate the influence of zymolyase treatment which is used for degradation of yeast cell wall. Two types of RNA extraction methods were used, and zymolyase preprocessing was applied to one among them. The qualities of total RNA extracted by two extraction methods were checked, and then RNAs were used for generating cloned DNA and RNA. Finally, two types of cRNA were hybridized to Affymetrix yeast genome 2.0_array. Keywords: comparison of RNA extraction process
2010-03-31 | GSE18456 | GEO
Project description:Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types
Project description:Recent advances in (meta)genomic methods have provided new opportunities to examine host-microbe-environment interactions in the human gut. While opportunities exist to extract DNA from freshly sourced colonic tissue there are potentially valuable sources of DNA from historical studies that might also be examined. We examined how four different tissue DNA extraction methods employed in past clinical trials might impact the recovery of microbial DNA from a colonic tissue sample as assessed using a custom designed phylogenetic microarray for human gut bacteria and archaebacteria. While all methods of DNA extraction produced similar phylogenetic profiles some extraction specific biases were also observed. Real time PCR analysis targeting several bacterial groups substantiated this observation. These data suggest that while the efficacy of different DNA extraction methods differs somewhat all the methods tested produce an accurate representation of microbial diversity. This suggests that DNA samples archived in biobanks should be suitable for retrospective analyses. Three technical replicates per sample (extraction method) were analysed
Project description:In daily practice biomolecules are usually extracted with their own specific protocols for downstream omics analysis. However, when sample material is limited, an all-in-one strategy is preferable. DNA, RNA and proteins can be isolated with phenol guanidine thiocyanate based extraction, yet lysis with Urea is the accepted standard for phosphoproteomic applications. Here we compared Urea with RNA-Bee mediated protein extraction for use in mass spectrometry (MS) based phosphoproteomics analysis of cells and tissues. We profile the DNA damage response after ionizing irradiation of U2OS cells as proof of principle for cultured human cells as well as mouse liver and three different human tissues. We show high overlap and similarity of phosphosite data and kinase activity for RNA-bee extraction when compared to standard Urea approach. Phenol guanidine thiocyanate lysis might thus be an appropriate way for multi-omics or MS profiling workflows in order to obtain several data types from a single sample.
Project description:This experiment aims to deepen our understanding of adipose tissue biology and its implications for metabolic and kidney health. The experimental workflow includes biopsy collection during surgery and total RNA extraction from each sample. Then cDNA libraries were prepared and used for high-throughput RNA sequencing.