Project description:Serum miRNAs are considered useful as non-invasive biomarkers for various diseases, but the optimal method for extracting RNA from serum is currently unknown. In this study, several RNA extraction kits were used to determine which kit is the optimal method. RNA was extracted from the serum of 8-week-old C57BL/6NJcl male mice according to the protocol of each RNA extraction kit. The yield of extracted RNA samples was calculated and electrophoretic patterns were evaluated by Agilent bioanalyzer. Expression patterns of the extracted RNA samples were confirmed by Agilent mouse miRNA microarray. The results showed significant differences in RNA yields in the miRNeasy serum/plasma advanced kit, and mirVana™ PARIS™ RNA and Native Protein Purification Kit compared to almost all other samples. Furthermore, two peaks were identified in the miRNeasy serum/plasma advanced kit using small RNA kit of Agilent bioanalyzer, one at 20-40 nucleotides (nt) and the other around 40-100 nt whereas the other reagents had a single peak. In addition, a high correlation was observed between the two RNA extraction kits in microarray. These results suggest that the above two kits are suitable for miRNA extraction from mouse serum.
Project description:Entamoeba histolytica membrane proteins are important players in the parasite’s pathogenicity. However, most of the proteins have not been identified. This study reports the membrane proteins extracted using three extractions methods: two commercial kits (ProteoExtract® from Calbiochem and ProteoPrep® from Sigma), and a conventional laboratory method. The resulting membrane fractions (MF) and cytosolic fractions (CF)were analysed using LC-ESI-MS/MS. The proteins identified in at least two out of three biological replicates revealed a total of 490, 492, and 587 MF proteins extracted using the ProteoExtract® kit, ProteoPrep® kit and conventional method, respectively. Meanwhile, 487, 611 and 343 proteins were identified in the CF extracted using the ProteoExtract® kit, ProteoPrep® kit and conventional method, respectively. Analysis of the identified MF and CF proteins extracted by the respective extraction kits suggests that the ProteoPrep® extraction kit was the most selective in separating MF and CF among the three extraction methods.
Project description:We present three different methods for protein extraction from fresh frozen mouse heart tissue: 1) extraction using SDS buffer followed by FASP purification, 2) extraction using SDS buffer followed by S-Trap purification, and 3) extraction using a guanidine hydrochloride buffer followed by in-solution digestion. Based on bicinchoninic acid assay, all three methods display similar recovery of total protein content. However, proteomics-based analysis identified far fewer proteins from the extraction guanidine hydrochloride. SDS-FASP identifies as many proteins as the SDS-STrap method with good coverage of proteins from different cellular compartments.
Project description:Effect of DNA extraction methods on the determination of the structure of microbial communities in the phosphogypsum waste heap soil
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:MicroRNAs (miRNA) are non-coding RNAs that negatively regulate gene expression by preventing the translation of specific mRNA transcripts. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits with and without addition of a carrier. We were able to profile miRNA levels in serum samples using the small RNA sequencing method on the Illumina platform and observed that successful sequencing cannot be predicted by substrate RNA quality. Although the carrier RNA had a significant impact on miRNA measurement, it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios and higher numbers of processed reads, but the majority of the reads were not aligning to miRBase. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies and by careful selection of an extraction method, permitting archived serum samples to become valuable resources for high-throughput applications.