Project description:In this study, we performed a comparative analysis of gut microbiota composition and gut microbiome-derived bacterial extracellular vesicles (bEVs) isolated from patients with solid tumours and healthy controls. After isolating bEVs from the faeces of solid tumour patients and healthy controls, we performed spectrometry analysis of their proteomes and next-generation sequencing (NGS) of the 16S gene. We also investigated the gut microbiomes of faeces from patientsand controls using 16S rRNA sequencing. Machine learning was used to classify the samples into patients and controls based on their bEVs and faecal microbiomes.
Project description:Gut microbial profiling of uterine fibroids (UFs) patients comparing control subjects. The gut microbiota was examined by 16S rRNA quantitative arrays and bioinformatics analysis. The goal was to reveal alterations in the gut microbiome of uterine fibroids patients.
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived TOX associated transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis. Methods: mRNA profiles of human CD8+ T cell overexpressed by TOX lentivirus or NC lentiviruswere generated by deep sequencing, in triplicate, using Illumina. qRT–PCR validation was performed using TaqMan and SYBR Green assays Conclusions: Our study represents the first detailed analysis of TOX associated transcriptomes generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
Project description:IL22 induces antimicrobial peptides which influnce microbiota. We used 16s rRNA gene sequencing (16s DNA-seq) to analyze the microbiota with Fc or IL-22Fc treatment.
Project description:Microbiota from rats fed with wheat aleurone and plant omega fatty acids In this study we investigated how an AX-rich WA and ALA from linseed oil (LO) modulate the gut microbiota of rats. Wistar rats were fed a standard diet and received either an iso-energetic control oil (PO), control oil + aleurone (A+PO), linseed oil (LO) or linseed oil + aleurone (A+LO) during 12 weeks. Feacal samples were recovered after the 12 week treatments. DNA extractions were performed using using the Qiagen's DNA Stool Kit (Qiagen, West Sussex, UK). 10ng of DNA template were amplified by PCR (16S gene) and purified using Qiagen's Qiaquick PCR purification kit (Qiagen, West Sussex, UK). 1ug of purified PCR product were labelled with either Cy3 or Cy5 using Genomic DNA ULS Labelling kit (Agilent Technologies, Palo Alto, CA). 250ng of labelled DNA were hybridized on the microarray for 24h at 65M-BM-0C. Washings were performed as recommended by the manufacturer. Microarray scanning was performed on a Surescan Microarray scanner (Agilent Technologies, Palo Alto, CA). Data were extracted using the Feature extraction software (Agilent Technologies, Palo Alto, CA). The retained intensity value for each probe was the ratio between the spotM-bM-^@M-^Ys median intensity signals and the median of background signals. A 13 chip study was realized to analyze the feacal microbiota of rats treated with either an iso-energetic control oil (PO), control oil + aleurone (A+PO), linseed oil (LO) or linseed oil + aleurone (A+LO) during 12 weeks. Each microarray corresponding to hybridization with 250ng of labelled 16S rRNA gene amplicons from 2 rat DNA faecal samples. Microbiota structure and diversity were assessed using the HuGChip (Tottey et al., 2013). Each probe (4441) was synthetized in three replicates. On the same array, 2 different samples were hybridized. One labelled with the Cy3 dye and one with the Cy5 dye. The results were processed as single channel (13 raw data files available on Series records for 25 samples).
Project description:Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived human M1- and M2-like macrophage profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to establish a high resolution transcriptome of human macrophages. Total RNA was isolated from classically and aternative activated human macrophages.mRNA profiles were generated by deep sequencing og M1 and M2 macrophages from 3 donors using Illumina HiSeqSQ. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Casava1.8 and TopHat followed by Cufflinks. qRT–PCR validation was performed using LightCycler and SYBR Green assays.
Project description:Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived human M1- and M2-like macrophage profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to establish a high resolution transcriptome of human macrophages. Total RNA was isolated from classically and aternative activated human macrophages.mRNA profiles were generated by deep sequencing og M1 and M2 macrophages from 3 donors using Illumina HiSeqSQ. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Casava1.8 and TopHat followed by Cufflinks. qRT–PCR validation was performed using LightCycler and SYBR Green assays. mRNA profiles were generated by deep sequencing og M1 and M2 macrophages from 3 donors using Illumina HiSeqSQ.