Small RNA sequencing-Illumina
Service Advantages
● the first base at the 5 'end has a strong preference for U to get specific RNAs
● miRNA target gene prediction
● The data is used to do joint analysis with mRNA
● miRNA differential expression
● miRNA target gene prediction
● BMKCloud-based result delivery: Customized data-mining available on platform
● After-sale services valid for 3 months upon project completion
Sample Requirements and Delivery
|
Library |
Platform |
Recommended data |
Data QC |
|
sRNA |
Illumina SE50 |
10M/20M reads |
Q30≥85% |
Sample Requirements:
Nucleotides:
|
Conc.(ng/μl) |
Amount (μg) |
Purity |
Integrity |
|
≥ 80 |
≥ 1 |
OD260/280=1.7-2.5 OD260/230=0.5-2.5 Limited or no protein or DNA contamination shown on gel. |
For plants: RIN≥7.5; For animals: RIN≥8.0; 5.0≥28S/18S≥1.0; limited or no baseline elevation |
*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
Experiment design
Sample delivery
RNA extraction
Library construction
Sequencing
Data analysis
After-sale services
Bioinformatics
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
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
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
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.
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
