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Small RNA sequencing-Illumina

Small RNA refers to a class of non-coding RNA molecules that are usually less than 200nt in length, including micro RNA (miRNA), small interference RNA (siRNA), and piwi-interacting RNA (piRNA).

MicroRNA (miRNA) is a class of endogenous small RNA with a length of about 20-24nt, which plays a variety of important regulatory roles in cells. miRNA involved in many life processes which revealing tissue – specific and stage – specific expression and highly conserved in different species.


Service Details

Bioinformatics

Demo Results

Case Study

Service Advantages

Ø the first base at the 5 'end has a strong preference for U to get specific RNAs
Ø miRNA target gene prediction
Ø The data is used to do joint analysis with mRNA
Ø miRNA differential expression
Ø miRNA target gene prediction
Ø BMKCloud-based result delivery: Customized data-mining available on platform.
Ø After-sale services valid for 3 months upon project completion

Sample Requirements and Delivery

Sample Requirements:

Nucleotides:

Purity Integrity Contamination Amount
OD260/280≥1.7-2.5; OD260/230≥0.5-2.5; For plants: RIN≥6.5; For animals: RIN≥7; 28S/18S≥1.0; limited or no baseline elevation Limited or no protein or DNA contamination shown on gel. Conc. ≥300 ng/μl; Volumn ≥ 10 μl; Total ≥ 3 μg

Tissue: Weight(dry): ≥1 g
*For tissue smaller than 5 mg, we recommend to send flash frozen(in liquid nitrogen) tissue sample.

Cell suspension: Cell count = 3×107
*We recommend to ship frozen cell lysate. In case that cell counts smaller than 5×105, flash frozen in liquid nitrogen is recommended.

Blood samples:
PA×geneBloodRNATube;
6mLTRIzol and 2mL blood(TRIzol:Blood=3:1)

Recommended Sample Delivery

Container:
2 ml centrifuge tube (Tin foil is not recommended)
Sample labeling: Group+replicate e.g. A1, A2, A3; B1, B2, B3... ...

Shipment:
1.Dry-ice: Samples need to be packed in bags and buried in dry-ice.
2.RNAstable tubes: RNA samples can be dried in RNA stabilization tube(e.g. RNAstable®) and shipped in room temperature.

Service Work Flow

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Experiment design

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Sample delivery

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RNA extraction

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Library construction

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Sequencing

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Data analysis

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After-sale services


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  • Bioinformatics

    CircRNA-sequencing-analysis-workflow

    1.miRNA Identification

    Each candidate precursor of novel miRNAs predicted by miRDeep2 has a pdf figure showing its structure and sequencing depth.

     miRNA-precursor-structure-and-sequencing-depth

    miRNA precursor structure and sequencing depth

    2.KEGG Enrichment of DE-miRNA Targeted Genes

    In this session, Biomarker firstly examined if the pathways are over-presentation with DE-miRNA targeted genes. Enrichment factors and fisher test were applied in the determination of enrichment degree and significance of the pathway. Equation of enrichment calculation is shown below.

    KEGG-pathway-enrichment-on-DE-miRNA-targeted-genes

    KEGG pathway enrichment on DE-miRNA targeted genes

    3.GO Enrichment Analysis on DE-miRNA Targeted Genes

    clusterProfiler was employed in GO enrichment analysis on DE-miRNA targeted genes. Directed acyclic graph of the enriched terms were generated to show the hierarchical structure of the terms. In the figure, the direction of arrows represents inclusion relations between terms, i.e. the nodes are more specific than their upper nodes. Directed acylic graph of DE-miRNA targeted genes was shown in the figure below.

    TopGO-directed-acyclic-graph-of-DE-miRNA-targeted-genes

    TopGO directed acyclic graph of DE-miRNA targeted genes

    BMK Case

    Integrated Analysis of MiRNA and Genes Associated with Meat Quality Reveals that Gga-MiR-140-5p Affects Intramuscular Fat Deposition in Chickens

    Published: Cellular Physiology and Biochemistry,2018

    Key results

    Late laying-period hens exhibited lower global expression levels of miRNAs than juvenile hens.
    A regulatory network of mirNA-mrna that may affect meat quality was constructed
    gga-miR-140-5p promotes adipocyte differentiation by down regulating RXRG.

    PB-full-length-RNA-Sequencing-case-study

    gga-miR-140-5p promotes chicken instramuscular preadipocyte differentiation by targeting the 3’UTR of RXRG

    Reference

    Zhang M ,  Li D H ,  Li F , et al. Integrated Analysis of MiRNA and Genes Associated with Meat Quality Reveals that Gga-MiR-140-5p Affects Intramuscular Fat Deposition in Chickens[J]. Cellular Physiology and Biochemistry, 2018:2421-2433. DOI: 10.1159/000489649

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