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Whole transcriptome sequencing – Illumina

Whole transcriptome sequencing is designed to profile all types of RNA molecules, including coding (mRNA) and non-coding RNAs (including lncRNA, circRNA and miRNA) which are transcribed by specific cells at a certain time. Whole transcriptome sequencing, also known as “total RNA sequencing” aims at revealing comprehensive regulatory networks at transcriptome level. Taking advantage of NGS technology, sequences of entire transcriptome products are available for ceRNA analysis and joint RNA analysis, which provides the first step towards functional characterization. Revealing regulatory network of circRNA-miRNA-mRNA based ceRNA.


Service Details

Bioinformatics

Demo Results

Case Study

Service Advantages

Ø Estimation on all types of RNAs in terms of counts, expression and chromosome-based relative expression
Ø Identification of differentially expressed RNAs and corresponding expression
Ø Gene co-expression analysis
Ø ceRNA network analysis
Ø Key genes involved pathway analysis
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. ≥100 ng/μl; Volumn ≥ 10 μl; Total ≥ 2 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.Estimation on all types of RNAs based relative expression
    Circos-on-the-significancy-of-the-differences-in-RNA-expression

    Circos on the significancy of the differences in RNA expression

    2.Integrated KEGG pathway network

    Integrated-KEGG-pathway-network

    Integrated KEGG pathway network

    3.ceRNA network analysis

    ceRNA-Network-based-DE-circRNA-miRNA-mRNA-Interactions

    ceRNA Network-based DE-circRNA-miRNA-mRNA Interactions

    BMK Case

    CircRNA Expression Pattern and ceRNA and miRNA–mRNA Networks Involved in Anther Development in the CMS Line of Brassica campestris

    Published: International Journal of Molecular Sciences,2019

    The NONMMUT015812-knockdown KP (shRNA-2) cells and negative control (sh-Scr) cells were obtained on day 6 of a specific viral infection.

    Key results

    This study established the Polima cytoplasm male sterility (CMS) line “Bcpol97-05A”, and the fertile line, “Bcajh97-01B”, in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis, and performed RNA expression profiling comparisons between the flower buds of the sterile line and fertile line by whole-transcriptome sequencing.
    1.A total of 31 differentially expressed (DE) circRNAs, 47 DE miRNAs, and 4779 DE mRNAs were identified.
    2.The gene ontology (GO) analysis demonstrated that Most of the DE genes were involved in pollen wall development
    3.The KEGG pathway enrichment analysis of the DE mRNAs (including the up-regulated anddown-regulated mRNAs in the sterile line compared to the fertile line) showed that “starch and sucrosemetabolism”, “phenylpropanoid biosynthesis”, and “pentose and glucuronate interconversions” werethe most enriched metabolic pathways.

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

    KEGG pathway enrichment analysis of the differentially expressed mRNAs

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

    Gene ontology (GO) classification of the differentially expressed mRNAs

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

    View of the DEcircRNA–DEmiRNA–DEmRNA triple network

    Reference

    Liang Y ,  Zhang Y ,  Xu L , et al. CircRNA Expression Pattern and ceRNA and miRNA–mRNA Networks Involved in Anther Development in the CMS Line of Brassica campestris[J]. International Journal of Molecular Sciences, 2019, 20(19):4808-. DOI: 10.3390/ijms20194808

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