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Small RNA sequencing

Arraystar Small RNA Profiling



Eukaryotic small RNAs are non-coding RNA molecules (ncRNA) less than 200 nt in length. The major small RNA biotypes include microRNA, tRF&tiRNA (tRNA derived RNA fragments and tRNA halves), sdRNA (sno-derived RNA), and piRNA, whose RNA sizes are tiny at less than 35 nt; and tRNA and snoRNA which are larger than 60 nt. Small RNAs are not random degradation products but rather RNA biotypes generated by precise biogenesis processes having unique biological features and functions. Small RNAs are commonly regulatory molecules fundamentally important in gene expression, epigenetics, genomics, and in diseases. Small RNAs are often plentiful.  Their abundance, stability and specific expression in the cells and in biofluids make them excellent candidates for biomarker applications.

Arraystar has long been focused on ncRNAs, such as lncRNA and circular RNA. Now, we continue the quest into the realm of small ncRNAs, which include not only the more studied microRNA, but also tRF/tiRNA, sdRNA, piRNA, tRNA, and snoRNA biotypes. To accelerate the analysis and the discovery, Arraystar has a broad portfolio of small RNA profiling services using nextgen sequencing platforms. To obtain the upmost quality, the small RNA profiling has been perfected specifically for the characteristic properties of that biotype, from sample pretreatment, experimental procedures, sequencing library preparation, and sequencing run. Most importantly, the dedicated annotations and data analyses tailored for each biotype are key to the understanding of their biology.

• Arraystar miRNA-seq
• Arraystar piRNA-Seq
• Arraystar tRF & tiRNA-Seq
• Arraystar tRNA-Seq

Arraystar RNA biotype-specific profiling strategy has unmatched performance, as regular small RNA-seq on mixed biotype pools is often insufficient for the coverage (Fig. 1) and the generic data analysis is inadequate for many of the RNA biotypes. Additionally, Arraystar microarray and  qPCR panel technologies for small RNAs are well established, ready to complement and maximize your capability in small RNA research.


Figure 1. piRNA and miRNA have a small difference in size distribution on the PAGE (A). Sequencing one size fraction for both miRNA and piRNA will significantly displace sequencing reads from each other, which is particularly problematic for very diverse piRNA sequences. The read counts for other small ncRNA biotypes are less than 2%, which are inadequate for their profiling (B).  Arraystar small RNA sequencing profiling effectively ensures biotype-specific coverage. 

miRNAs are the more studied small RNAs for their RNA interference functions in gene regulation and for biomarker uses. Arraystar miRNA-Seq size selects 15~35 nt for miRNA sequencing library preparation (Fig. 2). Arraystar miRNA annotation and the analysis package has gone beyond typical standards to include many advanced features. The single-base resolution covers the entire lengths of miRNAs for unambiguous identification of all microRNA isoforms (isomiR). The sequencing reads are always mapped with the latest reference database and novel miRNAs are discovered with the discovery pipeline. In addition to expression profiling and differential expression analysis, miRNA targets are predicted, gene regulatory network constructed, and gene ontology and pathways analyzed (Fig. 3).


Figure 2. miRNA library is sized for the best sequencing read coverage of microRNAs.

4.1Figure 3. Standard miRNA analysis package with advanced features.  (A) Novel miRNA discovery and pre-miRNA secondary structure prediction; (B) miRNA target prediction; (C) Gene ontology and pathway analysis.
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piRNAs are the largest class of diverse small non-coding RNAs of about 26-32 nucleotides in length, related to but distinct from miRNAs. piRNAs interact with the Piwi subfamily of Argonaute proteins. In contrast to several hundred microRNA species, tens of thousands of unique piRNA sequences are known in human, mouse and rat.  piRNAs are strikingly different from microRNAs in their length, expression pattern, genomic organization and biogenesis. Unlike miRNAs and siRNAs, piRNAs are not generated from dsRNA precursors by Dicer. Rather, piRNAs are produced from a primary transcript that traverses an entire piRNA cluster,  subsequently processed by phased Zucchini and amplified by Ping-pong cycles.
Functionally, piRNAs are important for transposon silencing to preserve genomic integrity during germline development and spermatogenesis. They are also involved in epigenetic regulation through histone modification, DNA methylation and heterochromatin assembly; regulation of translation and mRNA turn-over; maintenance of chromatin structure and cell cycle progression; and association with cancers. Many details and the exact function of piRNAs still remain open questions ripe for further study.

For studies interested in both miRNA and piRNA, piRNA-Seq is carried out in the same miRNA-Sequencing experiment, co-analyzed with microRNAs by including piRNA mapping, annotation and analyses.
For piRNA only sequencing, the precise size fraction for the piRNA population is recovered (Fig. 1).

For cells and tissues having  lower piRNA abundance, RNA samples are enriched for piRNAs by periodate oxidation and β-elimination (PO treatment), which efficiently removes non-piRNAs that do not have the 2’-O-Methylation protection at the 3’-end.

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Transfer RNAs (tRNAs) are ubiquitous and the most abundant of all small non-coding RNA molecules. As a fundamental component in translation, tRNAs serve as the physical link between the mRNA coding and protein sequences. A wide variety of biological processes, such as cell proliferation, differentiation and apoptosis, are always accompanied with variation of tRNAs levels. Alterations of tRNA repertoire affect cell-fate choices during cell development (Fig. 4). Dysregulated tRNA repertoire can promote tumorigenesis and cancer progression. Additionally, various diseases show disruptions to the levels and distributions of tRNAs, such as type 2 diabetes mellitus, Huntington disease, and HIV infection. Studying tRNA repertoire has become an important part of research of biological processes and human diseases. 


Figure 4. The tRNA repertoire determines the cell fate of proliferation or differentiation.

tRNAs undergo by far the greatest number of and the most chemically diverse post-transcriptional modifications, which is essential for tRNA stability, folding and decoding. As the amino acid carrier for peptide synthesis, tRNAs must be charged with amino acid. However, these aminoacylated termini and internal modifications impede the adaptor ligation and reverse transcription during tRNA-seq library preparation (Fig. 5). Scientists at Arraystar have developed state of the art tRNA-seq methodologies that integrate modification removal and small RNA sequencing optimized for tRNA, ensuring the most reliable and accurate tRNA-seq data for tRNA study.

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tRF & tiRNA-Seq
tRFs and tiRNAs, generated through precise biogenesis processes from tRNA (Fig. 5), are a major class of small RNAs, in many cases more abundant than microRNAs.  tRFs & tiRNAs perform many regulatory functions and are associated with many diseases and biological conditions. They are known to act as microRNAs in RNA interference; bind protein factors to regulate target mRNA stability; interact with cytochrome c to modulate apoptosis; alter embryonic transcriptional cascades as paternal epigenetic factor in intergenerational inheritance of metabolic disorders; and assemble stress granules in response to stress conditions (Fig. 7). The cell type/disease dependent composition and the highly enriched abundance in biofluids support the use of tRF&tiRNA in biomarker applications.
tRF & tiRNA-Seq, like tRNAs, are extensively modified by chemical groups at the 5’-, 3’- ends and in the internal sequence regions (Fig. 6).  These modifications impede efficient adapter ligation and reverse transcription, which until now presented significant challenges for sequencing in the past. With the expertise in tRF&tiRNA, Arraystar’s unique tRFs&tiRNA sequencing service offers:

 Breakthrough RNA pretreatments to remove modifications impeding successful sequencing
 Performance optimized tRFs&tiRNAs sequencing methodology
 Precise annotation and classification system based on tRNA topology and statistical significance.
 Comprehensive tRF & tiRNA collection from all databases.
 tRFs&tiRNAs focused bioinformatics and statistics analyses
 Included miRNA expression and differential analyses
 Seamless integration with our tRNA series of technologies: tRF&tiRNA-seq, tRNA-seq, tRF&tiRNA PCR array, and tRNA PCR array

Figure 5. Specific cleavages of mature or precursor tRNAs by angiogenin, Dicer, and other nucleases generate small RNA biotypes of tRFs and tiRNAs (tRNA halves).


Figure 6. Extensive modifications of tRFs, tiRNAs, and tRNAs prevent adapter ligation and reverse transcription during sequencing library construction. RNA pretreatments are essential for successful sequencing library reparation and profiling of these small RNAs.


Figure 7.  tRF&tiRNA functions and association with diseases.

Small nucleolar RNAs (snoRNAs) are involved in rRNA processing and regulation of splicing, translation, and oxidative stress (Williams & Farzaneh 2012).  Within snoRNPs, snoRNAs guide the 2'-O-methylation of rRNA by  C/D box snoRNA or  pseudouridylation by H/ACA box snoRNA. Additionally, snoRNAs can give rise to shorter miRNA-like sdRNAs capable of RNA interference (Ender et al., 2008). The dysregulated expression and roles in cancers and neurodegenerative diseases have been well documented (Nallar & Kalvakolanu 2013). SnoRNAs are relatively stable and can be detected in blood plasma, sputum, and urine samples, presenting a promising target for biomarker applications.

sno-derived RNAs (sdRNA) are produced from small nucleolar RNAs (snoRNA) either as H/ACA sdRNAs (20~24 nt) or C/D sdRNAs (bimodal distribution of 17~19 nt and around 30 nt), which are similar to the miRNA sizes (Fig. 8). sdRNAs can bind to Ago proteins (Ago1-4) and suppress  reporter genes having the sdRNA target sequences at 3’UTR. Moreover, some miRNAs annotated in miRBase are in fact sdRNAs. The majority of H/ACA sdRNAs are not generated by Drosha and DGCR8, but rather by spliceosomes and, in some cases, exonucleases. Dicer, however, is the endonuclease that processes into mature H/ACA sdRNA. The biogenesis of C/D sdRNAs is less known but seems independent of Drosha/DGCR8 and Dicer, which may include both specific  processing and non-specific degradation to account for the more diverse fragmentation. Larger sdRNAs may regulate alternative RNA splicing. sdRNAs display stronger differential expression than microRNAs and are massively upregulated in prostate cancer. Furthermore, the expression of some sdRNAs and their precursors is associated with clinical progression and metastatic occurrence.

sdRNAs are profiled identically as the tRF/tiRNA sequencing experiment, with raw sequencing reads mapped, annotated and analyzed for sdRNAs.


Figure 8. Some sdRNAs (shaded in color) derived from the snoRNAs.

Arraystar Small RNA Sequencing uses dedicated workflows for profiling tRNAs, tRF & tiRNA, miRNA, piRNA, and snoRNA classes with their own properties. Annotations and analyses are performed with the detailed information about small RNA biotype to gain biological insights.


* Basic annotation and analysis only
** Unmapped raw reads only. Analysis not included in the standard service package

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