Small RNA Sequencing

Service Overview

Small RNAs are type of short non-coding RNA with average length smaller than 200 nt. These small RNAs have been largely proved to play a pivotal role in various biological processes including development, diseases, metabolism, etc. Illumina enables in-depth sequencing and quantification of small RNA in cells, tissues, etc.

Service Workflow

Sample delivery
Library construction and sequencing
Data analysis
Final report
After-sale technical support

Bioinformatic Analysis

Results Demo

miRNA identification

miRNA transcription start sites are more frequently found in intergenic regions, introns and reverse strand of coding regions. miRNA genes are firstly transcribed into primary miRNA (pri-miRNA) and processed into precursor miRNA (pre-miRNA) and finally matured into miRNA with help of Dicer/DCL enzyme. Known miRNAs are identified by comparing mapped reads (against reference genome) with mature miRNA in miRBase(v21) database. Novel miRNAs are predicted by miRDeep2 software.

Small RNA base editing analysis

miRNA could be edited after transcription, resulting in change in seed sequence. As a consequence, its target gene will change.

miRNA expression quantification

RNA-Seq is able to achieve a highly sensitive estimation of gene expression. FPKM density curve and box plots can present the gene expression dispersion of a single sample by the mean time compared the overall expression level of different samples.

Hierarchical clustering of differentially expressed miRNA

Hierarchical cluster analysis enables clustering of miRNAs which share same pattern of expression across different experimental conditions. The genes within the same cluster are more likely to have similar biological functions and be involved in the same functional pathway. Therefore, it facilitates in-depth functional analysis on differentially expressed genes. In this analysis, differentially expressed miRNA will be clustered based on their expression pattern across different groups (experimental conditions).

Functional analysis on targeted genes of differentially expressed miRNA

In order to identify potential biological functions of the differentially expressed miRNA, their targeted genes are identified and processed for functional analysis including annotation and enrichment analysis.


1Are there any differences between animal and plant in miRNA target gene prediction?
Answer: Plant miRNAs target on coding region of genes, which result in binding to mRNA and lead to mRNA degradation. Therefore, the targeted genes could be predicted by mRNA sequence directly. In animals, miRNAs normally target on 3'UTR region of mRNA and inhibit translation of targeted gene. Therefore, comparing miRNA sequences directly to gene sequence won't identify targeted genes. Normally, 2000 bp before the 5' end of mRNA will be used for targeted gene prediction.
2How to find out the number of miRNA and corresponding pre-miRNA? (In some publications, they have phrases like “ We identified 83 potential novel miRNAs, which corresponded to 95 genomic loci.)
Answer: Some mature miRNA could originated from different pre-miRNAs. In non-reference based analysis, novel miRNAs are analyzed against and named after Unigenes. Repeated miRNAs are not counted. Therefore, by counting unique mature miRNA, conclusions can be made on the number of miRNA and that of corresponding pre-miRNA. For example, “In current analysis. 229 miRNA were predicted, within which 6 miRNA were originated from two pre-miRNAs.” can be described as “ We identified 223 miRNA, which correspond to 229 pre-miRNA.”
3How to validate miRNA targeted genes.
Answer: (1) Quantify miRNA by RT-PCR (2) miRNA over express (3) Process Luciferase assay on targeted gene of over-expressed miRNA. The fluorescent signal will be decreased if the gene expression is inhibited. (4) Degradome sequencing.

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