Exosomal Small RNA and miRNA Sequencing Service

For cell supernatant, a volume of ≥30 ml is recommended. Serum or plasma should be at least 4 ml. If providing exosomal RNA for miRNA - seq, it should be ≥10 ng, and for general exosome RNA - seq, ≥20 ng is required. Bile samples need to be ≥5 ml, urine ≥20 ml, and cerebrospinal fluid, chest fluid, or ascites ≥30 ml. The RNA sample should be in RNase - free water, RNA stabilization reagent, or 10 mM Tris pH 8.0, with an OD A260/A280 ratio ≥1.8, A260/230 ratio ≥1.8, and a RIN ≥6. Ship the samples with dry ice. Also, it's better to choose plasma over serum for research as serum may have more platelet - derived vesicles during the clotting process.

A：Our exosomal small RNA sequencing service primarily includes the following steps:
1)Exosomes are isolated and purified from biological samples like serum, plasma, urine, or cell culture supernatants using methods such as ultra-centrifugation, ultrafiltration, or immune-affinity capture.
2)Total RNA, including miRNA, is extracted from the isolated exosomes, preferably using a small-RNA-specific extraction kit.
3)The integrity, purity, and concentration of the RNA are evaluated by methods like agarose gel electrophoresis and spectrophotometry.
4)The RNA is reverse-transcribed into cDNA, and sequencing libraries are constructed by adding adapters and performing PCR amplification.
5)High-throughput sequencing platforms like Illumina are used to sequence the constructed libraries.
6)The sequencing data is subjected to quality control and pre-processing, such as removing low-quality reads, aligning the reads to the reference genome, identifying miRNAs, determining their expression levels, conducting differential expression analysis, and performing functional annotation and pathway analysis.

Due to the immunophenotyping of exosomes, the expression of relevant markers in the sample may be low, resulting in undetectable bands by WB. The characteristics of exosomes determine that not all items can be effectively detected by WB, especially when dealing with complex cell supernatants or low-yield plasma samples. It is generally recommended to select 2-3 positive markers and 1 negative marker for detection. Low positive rates are usually related to the sample itself.

The analysis results include sRNA annotation (classification statistics, length analysis), miRNA mature body identification (miRNA quantity and distribution, known miRNA species distribution, miRNA length distribution, base preference analysis), miRNA expression distribution (miRNA expression distribution in each sample, expression correlation scatter plot, sample-to-sample correlation analysis), miRNA differential expression analysis (up-and down-regulation statistical charts, clustering analysis diagrams, volcano plots, Venn diagrams), and target gene enrichment analysis (GO enrichment analysis, KEGG enrichment analysis).

Commonly used miRNA databases include miRBase and PMRD. These databases not only support miRNA retrieval but also can predict target genes through links to websites such as microRNAorg, TargetScan, and Pictar.