miRNA and piRNA are two biotypes of non-coding RNA molecules (ncRNA). The sizes of miRNA are tiny around 17nt to 25nt, and piRNA are tiny around 26nt to 32nt. As two kinds of small RNAs, they are not random degradation products but rather RNA biotypes generated by precise biogenesis processes having unique biological features and functions. They are commonly regulatory molecules fundamentally important in gene expression, epigenetics, genomics, and in diseases. 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 microRNA and piRNA biotypes. Arraystar has a qualified portfolio of miRNA and piRNA profiling services using nextgen sequencing platforms. Arraystar provides service 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

miRNA-Seq
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. 1). 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. 2).

Figure 1. miRNA library is sized for the best sequencing read coverage of microRNAs.

Figure 2. 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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piRNA-Seq
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.

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