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Small RNA Sequencing

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To support the increasing research interest in small RNA, CD Genomics is offering the qualified small RNA sequencing service that covers novel small RNA discovery, mutation characterization, and expression profiling of small RNAs by leveraging of advanced NGS technologies and data analysis pipeline.

The Introduction of Small RNA Sequencing

Small RNA species generally include the most common and well-studied microRNA (miRNA), small interfering RNA (siRNA), and piwi-interacting RNA (piRNA), as well as other types of small RNA, such as small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA). Small RNA is a type of lowly abundant, short in length (<200 nt), non-protein-coding RNAs that lack polyadenylation. Small RNA populations can vary significantly among different tissue types and species. Generally, small RNAs are formed by fragmentation of longer RNA sequences with the help of dedicated sets of enzymes and other proteins.

Small RNAs act in gene silencing and post-transcriptional regulation of gene expression. However, small RNA is not sufficient for the induction of RNA inference. It generally needs to form the core of the RNA-protein complex known as RNA-induced silencing complex (RISC). siRNAs can cleave the mRNA in the middle of the mRNA-siRNA duplex, and the resulting mRNA halves are degraded by other cellular enzymes. Unlike the siRNA pathway, miRNA-mediated degradation is initiated by enzymatic removal of the mRNA polyA tail. piRNAs are essential for the development of germ cells. Small RNAs have been demonstrated to be involved in a number of biological processes including development, cell proliferation and differentiation, and apoptosis.

By taking advantage of tremendous output with unprecedented sensitivity and dynamic range, NGS can identify weakly expressed small RNAs as well as quantitatively reveal heterogeneity in length and sequence. NGS is a powerful tool for investigating the function of small RNAs and prediction of potential mRNA target molecules without requiring available reference genomes. Obtaining a premium small RNA sequencing library begins with the isolation of small RNAs by size fractionation using gel electrophoresis selection or silica spin columns from total RNA. Following RNA adapter ligation using a 5’ adenylated DNA adapter with a blocked 3’end, small RNAs are reverse transcribed, amplified by PCR and sequenced. To identify and annotate known miRNAs, the sequencing reads can be mapped to a species-specific database, such as mirWalk and miRBase.

Advantages of Small RNA Sequencing

  • Small RNA and miRNA profiling
  • Understanding how post-transcriptional regulation contributes to the phenotype
  • Identifying more unmapped small RNAs and isoforms, as well as novel biomarkers

Small RNA Sequencing Workflow

CD Genomics utilizes the Illumina HiSeq platforms to sequence small RNAs. We have flexible strategies for miRNA (15-30nt) and/or small RNA (30-200nt) discovery and profiling. Our highly experienced expert team executes quality management, following every procedure to ensure confident and unbiased results. The general workflow for small RNA sequencing is outlined below.

Service Specification

Sample requirements and preparation

  • Sample type: Total RNA without degradation or DNA contamination
  • Starting amount of total RNA ≥ 5 µg
  • Sample concentration ≥ 200 ng/µl
  • Sample purity: OD260/280 = 1.8~2.2
  • All RNA samples are validated for purity and quantity
Sequencing

  • Libraries of miRNA (15-30nt), small RNA (30-200nt), or custom size range
  • Illumina HiSeq SE50
  • ≥ 10 M reads
  • More than 90% of bases with a ≥Q30 quality score
Bioinformatics Analysis
We provide customized bioinformatics analysis including:

  • Raw data quality control and length filter
  • Reference-based mapping
  • Small RNA classification and quantification
  • Target gene prediction and annotation
  • Novel small RNA prediction
  • Target gene analysis of differentially expressed small RNA (GO enrichment and KEGG enrichment)

Analysis pipeline

CD Genomics provides full small RNA sequencing service packages including sample standardization, library preparation, deep sequencing, raw data quality control, genome assembly, and customized bioinformatics analysis. We can tailor this pipeline to your research interest. If you have additional requirements or questions, please feel free to contact us.

Related Services
RNA-Seq

Transcriptome mapping and quantification

LncRNA Sequencing

Expression profiling of long non-coding RNAs

CircRNA Sequencing

Expression profiling of circular RNAs

Degradome Sequencing

Insight into microRNAs landscape

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45-1 Ramsey Road, Shirley, NY 11967, USA
Tel: 1-631-275-3058
Fax: 1-631-614-7828
Email: info@cd-genomics.com