Introduction
microRNAs (miRNAs) are 18-25 nt single-stranded RNAs (ssRNAs) which are generated from endogenous hairpin-shaped transcripts. miRNAs function as guide molecules in post-transcriptional gene silencing by base pairing with target mRNAs, which leads to mRNA cleavage or translational repression. Numerous studies suggest that miRNA-mediated silencing may play an important role in development and disease, and implies that miRNA can serve as valuable biomarkers for diagnostic approaches.
Historically, the methods employed for characterizing miRNA have been computational prediction, qPCR, and microarray hybridization. These methods focus primarily on miRNA quantification and are limited to studying miRNA with previous sequence information or secondary stem loop structures. The high throughput sequencing technique has demonstrated to be of a great benefit when identifying and profiling miRNAs. Direct sequencing offers the potential to detect variation in mature miRNA lengths, as well as enzymatic modifications of miRNAs such as RNA editing and 3' nucleotide additions. This provides a more complete view of the miRNA transcriptome. It also offers an opportunity to identify low-abundance miRNAs or those miRNAs exhibiting modest expression differences between samples, which may be functional, but cannot be detected by hybridization-based methods.
Benefits of microRNA Sequencing
Technology
• Highly accurate base-by-base sequencing eliminates cross-hybridizations
• The count numbers of individual transcripts are used to measure the abundance of microRNA expression abundance
Coverage
• Does not depend on any prior sequence information and provides information about all microRNAs within the sample which allows for the discovery of novel microRNAs
Annotation
• microRNA annotation always refers to the latest miRBase
Specificity
Specificity
• Capable of discriminating mature microRNA from pre-microRNA
Reproducibility
• Allows for the detection of microRNAs with a low abundance and small alterations while maintaining excellent reproducibility
Arraystar offers high throughput microRNA sequencing services using the Illumina sequencing platform. Our full sequencing service includes optimized microRNA sequencing library construction, high throughput microRNA sequencing, and Superior Data Analysis.
Recent Publications Citing Our microRNA Sequencing Service
Molecular fingerprinting of the podocyte reveals novel gene and protein regulatory networks.
Melanie Boerries, et al. Kidney International. 2013
Description of Services
Please refer to Sample Submission for details in how to get your project started.
2. Sequencing library preparation
3. Cluster Generation on Cluster Station
4. Sequencing by Illumina platform
5. Data extraction, analysis and summarization
Highlight Features of Arraystar's microRNA Sequencing Service
• Low RNA Input Requirements
• Analysis of isomiRs
• Differential Expression Analysis Using Three-way Read Counts
• Differential Expression Analysis Using Three-way Read Counts
• Detection of miRNAs with a Low-abundance
• Cost-effective Full Service from Sample to Data
Low RNA Input Requirements
Low RNA Input Requirements
Requires 1.0 ug or less of total RNA for analysis of precious samples with high sensitivity, detecting as low as a single copy per cell.
Analysis of isomiRs
The deep sequencing of miRNAs will identify a diverse population of variants of known miRNAs, which are hardly to be detected by hybridization based methods. It is reported that the expression of these variants and miR, may increase the complexity of miRNA processing and regulation.
The deep sequencing of miRNAs will identify a diverse population of variants of known miRNAs, which are hardly to be detected by hybridization based methods. It is reported that the expression of these variants and miR, may increase the complexity of miRNA processing and regulation.
IsomiRs: miRNAs frequently exhibit variation from their reference sequences, producing multiple mature variants that we refer to as isomiRs. The difference in sequence between the variants is primarily found at the 3' end of the molecule, sometimes at 5'end. The functional significance of this heterogeneity is still not fully understood. It appears that much of the isomiR variability can be explained by variability in either, Dicer1 or Drosha, cleavage positions within the pre-miRNA hairpin.
Differential Expression Analysis Using Three-way Read Counts
The absolute number of sequence reads for a particular microRNA represents a measure of its relative abundance. Because of the existence of isomiR, choosing a different isomiR sequence to measure miRNA expression levels can affect the ability to detect differentially expressed miRNA. We provide three different counting methods for miRNA expression level by using the copy number of the most abundant isomiRs, all isomiRs and the miRBase sequences (Table 1). Thus, the researcher can choose a suitable explanation of the expression levels of certain miRNA according to their different research interests. However, it is reported that the read count for the most abundant isomiR, rather than the miRBase reference sequence, provides the most robust approach for comparing miRNA expression between samples.
|
|
Most_Abundant_ |
Fold Change |
ALL_ IsomiRs (TPM*) |
Fold Change |
miRBase_seq (TPM*) |
Fold Change | |||
|
MATURE-ID |
Con. |
Test |
Test Vs. Con. |
Con. |
Test |
Test Vs. Con. |
Con. |
Test |
Test Vs. Con. |
|
hsa-miR-1 |
11 |
685 |
0.0302158 |
47 |
1699 |
0.0333528 |
11 |
685 |
0.0302158 |
|
hsa-miR-2 |
208 |
10 |
10.9 |
257 |
10 |
13.35 |
208 |
10 |
10.9 |
|
hsa-miR-3 |
117 |
71 |
0.637795 |
260 |
123 |
0.492593 |
63 |
26 |
0.493151 |
Table 1. Differential expression levels calculated using three-way read counts.
Low-abundance miRNAs
microRNA sequencing also provides the opportunity to identify low-abundance microRNAs. It has been reported that there is some correlation between the evolutionary conservation of miRNAs and their expression levels. Evolutionarily conserved miRNAs were often among the most abundant miRNAs while some low-abundance non-conserved miRNAs may be newly evolved and be important to specific species. These low-abundance miRNAs also may be observed in higher quantities among some specific tissues where they are functionally important.
Reference
1. Morin, R.D., et al., Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. Genome Res, 2008. 18(4): p. 610-21.
2. Glazov, E.A., et al., A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach. Genome Res, 2008. 18(6): p. 957-64.
3. Jagadeeswaran, G., et al., Deep sequencing of small RNA libraries reveals dynamic regulation of conserved and novel microRNAs and microRNA-stars during silkworm development. BMC Genomics, 2010. 11: p. 52.
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