Project description:Exosomes, endosome-derived membrane microvesicles, contain a specific set of RNA transcripts that are involved in cell-cell communication and hold a great potential as disease biomarkers. To systemically characterize exosomal RNA profiles, we performed RNA sequencing analysis using three human plasma samples and evaluated efficacies of small RNA library preparation protocols from 3 manufacturers. We tested the six samples (A1 and A2, B1 and B2, C1 and C2) using two small RNA library preparation kits: NEBNext Multiplex Small RNA library Prep Set from New England Biolab (NEB) and NEXTflex Small RNA Sequencing Kit from Bioo Scientific (BS). We also tested IlluminaM-bM-^@M-^Ys TrueSeq Small RNA Sample Preparation Kit (ILMN) in sample A1 and A2. Together, we tested these plasma samples by sequencing 14 indexed libraries. This study allowed direct comparison of current small RNA library preparation protocols and identified the most suitable strategy for future exosomal RNA sequencing analysis.
Project description:RNA-sequencing (RNA-Seq) protocols and bioinformatic pipelines are designed to streamline downstream analyses on sequences believed to be the most important. Here, we have challenged this dogma by preserving ribosomal RNA (rRNA) in our samples and by lowering the minimal RNA size window of our small RNA-Seq analyses to 8 nt
Project description:Exosomes, endosome-derived membrane microvesicles, contain a specific set of RNA transcripts that are involved in cell-cell communication and hold a great potential as disease biomarkers. To systemically characterize exosomal RNA profiles, we performed RNA sequencing analysis using three human plasma samples and evaluated efficacies of small RNA library preparation protocols from 3 manufacturers.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:To identify the candidate exosomal miRNAs involved in ESCC metastasis, we conducted small RNA sequencing to determine the miRNA expression profiles of plasma exosomes from 5 LNM+ and 5 LNM- ESCC patients
Project description:Purpose: Analysis of exosomal miRNA in plasma of patients with metastatic and non-metastatic pancreatic cancer. To identify exosomes cargo specific miRNAs promoting cancer metastasis. Methods: We divide the plasma samples into five groups according to whether they have metastasis and undergo surgery.The five groups are Health(20),Metastasis Pre-operation and Post-operation (14),Non-metastasis Pre-operation and Post-operation(9).Then we collected the exosomes by ultracentrifugation and extracted the small RNA in each sample.Then, we analyzed the expression changes of miRNA by small RNA sequencing. Result: Different groups have different expression of small RNA. Compared with the non-metastatic group, the expression of miR-92a-3p , miR-148-3p and miR-25-3p increased in the metastatic group. Conclusion:Exosomal miRNA in pancreatic cancer patient derived plasma were analyze using Hiseq2500 sequencing technique. We identified that miR-92a-3p, miR-148-3p and miR-25-3p enriched in metastatic pancreatic cancer patient plasma derived exosomes.