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:This is for Rigor and Reproducibility study to demonstrate the reproducibility of AFS SOP for EV separation, RNA isolation, and RNA analysis by using saliva samples from healthy individuals. Blinded saliva samples have been processed by two independent AFS operators for EV separation and subjected to miRNA-seq analysis. EV-associated miRNA seqeunces were used as a monitoring tool for Rigor and Reproducibility of AFS process between operators.
Project description:Background Ribosome depleted RNA-seq is well suited for understanding coding- and noncoding transcript expression in clinical samples with diverse input RNA quality and mass. Current choices for RNA extraction include phase-separation (e.g., ThermoFisher TRIzol) approaches to column-based (e.g., Norgen Spin Columns) approaches. Each method, however, has been noted to have differing effects on the properties of the extracted nucleic acids, precluding the straightforward bioinformatic comparison of data generated by the two methods. Methods In this study, we present differences in data differing only in total RNA extraction approaches (phase separation vs. column) from clinical samples processed by the same tissue bank and sequencing facility personnel. We use data from seven samples with libraries generated from RNA extracted with both extraction methods to characterize the effect of ‘differentially extracted’ genes (DExGs) on transcriptomic profiles. We then use another set of 148 samples extracted with one or the other method to confirm the differences observed in the 7-sample dataset. Results The large number of samples employed in this study allows us to find significant differences in important quality control parameters as well as many DExGs between the two methods. We identify several biophysical properties which are potential drivers of these differences. Conclusions We show that partial correction of extraction method mediated differences can be achieved but full correction is not possible. These results lay the foundation for principled decision making of RNA extraction methods for use in clinical samples in a translational environment, particularly in light of recent reagent shortages during the COVID-19 pandemic.
Project description:Purpose: This study aims to compare and analyze the differences in bacterial community composition in fecal samples from mice treated with Control(DW), Vancomycin (VAN), Ampicillin (AMP), Neomycin (NEO), Metronidazole (MET), and a combination of all antibiotics (ALL, VANM) using 16S rRNA sequencing. Methods: Each antibiotics treated mice's fecal samples were collected and stored -80'c until analyzation. DNA was extracted using the NucleoSpin DNA Stool Kit (MACHEREY-NAGEL) following the manufacturer’s protocol. Metagenomic sequencing was performed on an Illumina MiSeq platform (Illumina), targeting the V3 and V4 regions of the 16S rRNA gene according to the manufacturer's instructions. PCR products were purified using AMPure XP beads, and sequencing adapters were added using the Nextera XT Index Kit (Illumina). The library was further purified with AMPure XP beads and quantified using automated electrophoresis with the TapeStation System (Agilent). Sequencing was performed using the MiSeq v3 reagent kit (Illumina), following the manufacturer’s protocol. Results: QIIME2 (v2023.02) was used to process and analyze 16S rRNA gene amplicon sequencing data, from sequence preprocessing to taxonomic classification. Paired-end sequences were merged and quality-filtered using Deblur. The resulting amplicon sequence variants (ASVs) were used for downstream analyses. Conclusions: Our study presents a comparative analysis of bacterial community composition in fecal samples from antibiotic-treated mice. We observed that microbiota composition varied distinctly depending on the type of antibiotic administered.
Project description:In this study, we quantitated the disappearance of intact HMOs and characterized the glycan digestion products in the gut that are produced by the action of microbial enzymes on HMOs and glycoconjugates from breast milk. Oligosaccharides from fecal samples of exclusively breast-fed infants were extracted and profiled using nanoLC-MS. Intact HMOs were found in the fecal samples, additionally, other oligosaccharides were found corresponding to degraded HMOs and non-HMO based compounds. The latter compounds were fragments of N-glycans released through the cleavage of the linkage to the asparagine residue and through cleavage of the chitobiose core of the N-glycan.
Project description:The identification of putative biomarkers for predicting abdominal aortic aneurysm (AAA) progression can be achieved through proteomic strategies, particularly with shotgun proteomics. However, the heterogeneity of AAA tissue poses challenges for tissue homogenization and protein extraction, compromising reproducibility, an essential factor for clinical translation. Thus, we aimed to optimize a protocol for AAA tissue homogenization and protein extraction and to develop a standard operating procedure (SOP) to maximize protein yield and foster reproducibility in the field. In this context, several experimental variables were systematically tested in a bead-beating method for tissue homogenisation, including bead size (1.4 mm versus 2.8 mm of diameter), the number of extraction cycles (one up to three), a bead-to-tissue mass ratio at 20, 30, or 40, the lysis buffer-to-tissue ratio (10, 15, or 20 μL/mg), and the lysis buffer composition (RIPA, Urea/thiourea, and HEPES). Optimal conditions for protein extraction were achieved using 1.4 mm zirconium dioxide beads in two homogenization cycles, with a bead-to-tissue mass ratio of 30:1 and a lysis buffer-to-tissue ratio of 20 μL lysis buffer per milligram of tissue, in 2 mL O-ring cryotubes. Depending on the purpose of downstream analysis, recommendations for the use of each lysis buffers were provided based on their specific performance across specific indexes. In this context, HEPES and Urea/Thiourea buffers are recommended for studies prioritizing quantification reproducibility, while RIPA buffer is advised for protein extraction reproducibility. As a proof of concept, the optimized SOP was applied to characterize the AAA tissue proteome and several processes and pathways relevant in AAA pathophysiology were highlighted, including blood coagulation, cellular responses to stress, neutrophil degranulation, wound healing, and pathways of the immune system. Additionally, the enrichment analysis identified ECM remodeling and platelet activation, and degranulation as strongly represented pathways across the functional analysis for each buffer, reinforcing their contribution to aneurysm pathophysiology. Hence, these findings demonstrated that the SOP is well-suited for identifying disease-relevant biomarkers and therapeutic targets.
Project description:Total RNA was extracted from mouse liver samples using an Ambion RNaqueous RNA extraction kit. Targets were produced using modified Affymetrix procedure from 5ug of total RNA. 3 biological replicates were analysed per each group of animals. Keywords = growth hormone Keywords = signaling Keywords = gene expression Keywords: other
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.
Project description:A subset of post-infection irritable bowel syndrome (PI-IBS) patients have elevated, or high fecal proteolytic activity (PA). Fecal PA has been shown to correlate with increased symptom severity as well as lower quality of life scores, increased fecal output and increased intestinal permeability. To address the underlying mechanisms of barrier disruption as a consequence of high fecal PA, colonic biopsies were collected from healthy individuals PI-IBS patients (n=11). Individuals diagnosed with PI-IBS were further divided in to 2 subgroups, high PA and low PA as defined by the PA in matched fecal samples. RNA was extracted from the biopsies for bulk RNA sequencing to understand transcriptional differences between healthy and high PA PI-IBS patients as well as high PA and Low PA PI-IBS patients.