Small and microRNA Sequencing
microRNAs (miRNA) are a class of small non-coding RNAs typically 21-23 nt long found in plants, animals, and some viruses (Figure 1). miRNAs play a pivotal role in RNA silencing and post-transcriptional regulation of gene expression. Discovered in the early 1990s, miRNA research has revealed (i) multiple roles for miRNAs in development (ii) disease-associated aberrant expression of miRNAs and (iii) the importance of miRNA in many other biological processes. Next generation sequencing (NGS) technologies have become a powerful tool to study genome-wide miRNA expression patterns and have helped to identify disease associations, isoforms of miRNAs, and to discover previously uncharacterized miRNAs.
Experimental Design: As an expert in the area of miRNA-Seq, Microsynth is able to provide a one-stop service from experimental design consulting up to bioinformatics analysis. Should you not involve Microsynth in your experimental design, please consider the importance of the number of biological replicates. We usually advise to include at least 3 biological replicates per condition, to finally obtain statistical significance for your differential miRNA expression analysis.
RNA Isolation: Either you leave it up to Microsynth or you use a commercial kit to isolate total RNA used for the Illumina miRNA-Seq protocol.
Library Preparation and Sequencing: Following a quality check of your total RNA samples, Microsynth will perform miRNA enrichment. Illumina cDNA library is generated by reverse-transcription including specific sequencing adaptors with barcodes. Finally, the libraries are pooled and sequenced on the Illumina machine. The envisaged number of reads per library depends on the organism under study and the desired sensitivity. The usually required number of reads for higher eukaryotic species (e.g. human, rat, mouse) is approx. 5-15 Mio reads depending whether complex tissues or unique type of cells are analyzed.
Provided Output Files: See examples below
Figure 2. Exemplary extract of the comparison of cluster representative sequence (query id) against the miRBase database (subject id) using UBLAST. Clusters that show no significant blast hit can be regarded as putative novel miRNAs.
Figure 3. Exemplary extract of the de-novo motif search results. Given the p-value and the false discovery rate, motif 5 (*) is rated as putative false positive motif. Additionally total target and background sequences are listed and links to known motifs and gene ontology enrichment results are provided if present (not shown).
- siRNA synthesis service at Microsynth
- RNA-Sequencing at Microsynth
- CHiP Seq analysis pipeline at Microsynth
- Griffiths, J.S., (2004) The microRNA Registry. Nucl. Acids Res. 32 (suppl 1): D109-D111.
- Dobin et al. (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 29:15-21.
- Love et al (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15: 550.
- Edgar R.C. (2010) Search and clustering orders of magnitude faster than BLAST, Bioinformatics 26: 2460-2461.
- Heinz et al. (2010) Simple Combinations of Lineage-Determining Transcription Factors Prime cis-Regulatory Elements Required for Macrophage and B Cell Identities. Mol Cell 38: 576-589.
- Mestdagh et al. (2014) Evaluation of quantitative miRNA expression platforms in the microRNRNA quality control (miRQC) study. Nat Methods. 11: 809-815.