A Review of Discovery Profiling of PIWI-Interacting RNAs and Their Diverse Functions in Metazoans.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs (sncRNAs) that perform crucial biological functions in metazoans and defend against transposable elements (TEs) in germ lines. Recently, ubiquitously expressed piRNAs were discovered in soma and germ lines using small RNA sequencing (sRNA-seq) in humans and animals, providing new insights into the diverse functions of piRNAs. However, the role of piRNAs has not yet been fully elucidated, and sRNA-seq studies continue to reveal different piRNA activities in the genome. In this review, we summarize a set of simplified processes for piRNA analysis in order to provide a useful guide for researchers to perform piRNA research suitable for their study objectives. These processes can help expand the functional research on piRNAs from previously reported sRNA-seq results in metazoans. Ubiquitously expressed piRNAs have been discovered in the soma and germ lines in Annelida, Cnidaria, Echinodermata, Crustacea, Arthropoda, and Mollusca, but they are limited to germ lines in Chordata. The roles of piRNAs in TE silencing, gene expression regulation, epigenetic regulation, embryonic development, immune response, and associated diseases will continue to be discovered via sRNA-seq.

Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess Their Role in RNA Spray-Based Crop Protection.

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5'-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.

A virus-derived siRNA activates plant immunity by interfering with ROS scavenging.

Virus-derived small interference RNAs (vsiRNAs) not only suppress virus infection in plants via induction of RNA silencing but also enhance virus infection by regulating host defensive gene expression. However, the underlying mechanisms that control vsiRNA-mediated host immunity or susceptibility remain largely unknown. In this study, we generated several transgenic wheat lines using four artificial microRNA expression vectors carrying vsiRNAs from Wheat yellow mosaic virus (WYMV) RNA1. Laboratory and field tests showed that two transgenic wheat lines expressing amiRNA1 were highly resistant to WYMV infection. Further analyses showed that vsiRNA1 could modulate the expression of a wheat thioredoxin-like gene (TaAAED1), which encodes a negative regulator of reactive oxygen species (ROS) production in the chloroplast. The function of TaAAED1 in ROS scavenging could be suppressed by vsiRNA1 in a dose-dependent manner. Furthermore, transgenic expression of amiRNA1 in wheat resulted in broad-spectrum disease resistance to Chinese wheat mosaic virus, Barley stripe mosaic virus, and Puccinia striiformis f. sp. tritici infection, suggesting that vsiRNA1 is involved in wheat immunity via ROS signaling. Collectively, these findings reveal a previously unidentified mechanism underlying the arms race between viruses and plants.

Genome-wide analysis of brassinosteroid responsive small RNAs in Arabidopsis thaliana.

BACKGROUND: Brassinosteroids (BRs) are a class of phytohormones with important roles in regulating physiological and developmental processes. Small RNAs, including small interfering RNAs and microRNAs (miRNAs), are non-protein coding RNAs that regulate gene expression at the transcriptional and post-transcriptional levels. However, the roles of small RNAs in BR response have not been studied well. OBJECTIVE: In this study, we aimed to identify BR-responsive small RNA clusters and miRNAs in Arabidopsis. In addition, the effect of BR-responsive small RNAs on their transcripts and target genes were examined. METHODS: Small RNA libraries were constructed from control and epibrassinolide-treated seedlings expressing wild-type BRI1-Flag protein under its native promoter in the bri1-5 mutant. After sequencing the small RNA libraries, differentially expressed small RNA clusters were identified by examining the expression levels of small RNAs in 100-nt bins of the Arabidopsis genome. To identify the BR-responsive miRNAs, the expression levels of all the annotated mature miRNAs, registered in miRBase, were analyzed. Previously published RNA-seq data were utilized to monitor the BR-responsive expression patterns of differentially expressed small RNA clusters and miRNA target genes. RESULTS: In results, 38 BR-responsive small RNA clusters, including 30 down-regulated and eight up-regulated clusters, were identified. These differentially expressed small RNA clusters were from miRNA loci, transposons, protein-coding genes, pseudogenes and others. Of these, a transgene, BRI1, accumulates small RNAs, which are not found in the wild type. Small RNAs in this transgene are up-regulated by BRs while BRI1 mRNA is down-regulated by BRs. By analyzing the expression patterns of mature miRNAs, we have identified BR-repressed miR398a-5p and BR-induced miR156g. Although miR398a-5p is down-regulated by BRs, its predicted targets were not responsive to BRs. However, SPL3, a target of BR-inducible miR156g, is down-regulated by BRs. CONCLUSION: BR-responsive small RNAs and miRNAs identified in this study will provide an insight into the role of small RNAs in BR responses in plants. Especially, we suggest that miR156g/SPL3 module might play a role in BR-mediated growth and development in Arabidopsis.

Extracellular vesicles from Heligmosomoides bakeri and Trichuris muris contain distinct microRNA families and small RNAs that could underpin different functions in the host.

Extracellular vesicles (EVs) have emerged as a ubiquitous component of helminth excretory-secretory products that can deliver parasite molecules to host cells to elicit immunomodulatory effects. RNAs are one type of cargo molecule that can underpin EV functions, hence there is extensive interest in characterising the RNAs that are present in EVs from different helminth species. Here we outline methods for identifying all of the small RNAs (sRNA) in helminth EVs and address how different methodologies may influence the sRNAs detected. We show that different EV purification methods introduce relatively little variation in the sRNAs that are detected, and that different RNA library preparation methods yielded larger differences. We compared the EV sRNAs in the gastrointestinal nematode Heligmosomoides bakeri with those in EVs from the distantly related gastrointestinal nematode Trichuris muris, and found that many of the sRNAs in both organisms derive from repetitive elements or intergenic regions. However, only in H. bakeri do these RNAs contain a 5' triphosphate, and Guanine (G) starting nucleotide, consistent with their biogenesis by RNA-dependent RNA polymerases (RdRPs). Distinct microRNA (miRNA) families are carried in EVs from each parasite, with H. bakeri EVs specific for miR-71, miR-49, miR-63, miR-259 and miR-240 gene families, and T. muris EVs specific for miR-1, miR-1822 and miR-252, and enriched for miR-59, miR-72 and miR-44 families, with the miR-9, miR-10, miR-80 and let-7 families abundant in both. We found a larger proportion of miRNA reads derive from the mouse host in T. muris EVs, compared with H. bakeri EVs. Our report underscores potential biases in the sRNAs sequenced based on library preparation methods, suggests specific nematode lineages have evolved distinct sRNA synthesis/export pathways, and highlights specific differences in EV miRNAs from H. bakeri and T. muris that may underpin functional adaptation to their host niches.

A universal method for the rapid isolation of all known classes of functional silencing small RNAs.

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.

Potential functional pathways of plant RNA virus-derived small RNAs in a vector insect.

During infection, RNA viruses can produce two types of virus-derived small RNAs (vsRNAs), small interfering RNA (siRNA) and microRNA (miRNA), that play a key role in RNA silencing-mediated antiviral mechanisms in various hosts by associating with different Argonaute (Ago) proteins. Ago1 has been widely identified as an essential part of the miRNA pathway, while Ago2 is required for the siRNA pathway. Thus, analysis of the interaction between vsRNAs and Ago proteins can provide a clue about which pathway the vsRNA may be involved in. In this study, using rice stripe virus (RSV)-small brown planthoppers (Laodelphax striatellus, Fallen) as an infection model, the interactions of eight vsRNAs derived from four viral genomic RNA fragments and Ago1 or Ago2 were detected via the RNA immunoprecipitation (RIP) method. vsRNA4-1 and vsRNA4-2 derived from RSV RNA4 were significantly enriched in Ago1-immunoprecipitated complexes, whereas vsRNA2-1 and vsRNA3-2 seemed enriched in Ago2-immunoprecipitated complexes. vsRNA1-2 and vsRNA2-2 were detected in both of the two Ago-immunoprecipitated complexes. In contrast, vsRNA1-1 and vsRNA3-1 did not accumulate in either Ago1- or Ago2-immunoprecipitated complexes, indicating that regulatory pathways other than miRNA or siRNA pathways might be employed. In addition, two conserved L. striatellus miRNAs were analysed via the RIP method. Both miRNAs accumulated in Ago1-immunoprecipitated complexes, which was consistent with previous studies, suggesting that our experimental system can be widely used. In conclusion, our study provides an accurate and convenient detection system to determine the potential pathway of vsRNAs, and this method may also be suitable for studying other sRNAs.

siRNA Library Screening to Identify Complementary Therapeutic Pairs in Triple-Negative Breast Cancer Cells.

The existence of tightly integrated cross talk through multiple signaling and effector pathways has been appreciated in malignant cells. The realization of the plasticity of such networks is stimulating the development of combinational therapy to overcome the limitations of one-dimensional therapies. Synergistic pairs of siRNAs or siRNA and drug combinations are the new frontiers in identifying effective therapeutic combinations. To elucidate effective combinations, we developed a versatile protocol to screen siRNA libraries in triple-negative breast cancer cell models. This protocol outlines the steps to identify synergistic combinations of siRNA-siRNA or siRNA-drug combinations using siRNA libraries via a robotic screen. By focusing on smaller functional siRNA libraries, we present methodologies to identify synergistic siRNA pairings against cancerous cell growth and molecular targets to augment the activity of pro-apoptotic TRAIL protein. Here, we summarize the critical steps to undertake such combinational target identification, emphasizing critical factors that affect the outcome of the screens. Our experience suggests that siRNA library screening is an efficient protocol to identify complementary therapeutic pairs of new or already-existing drugs. This protocol is simple, robust and can be completed within a 1-week working period.

In Vitro Evaluation of Candidate Gene Targets for Cancer Therapy.

Discovery and development of gene targets for cancer therapeutics are lengthy and highly costly processes. Identification and evaluation of candidate gene targets are of fundamental importance. RNA interference allows candidate genes to be specifically and effectively knocked down in cancer cells. This tool can be easily incorporated into a loss-of-function approach in the initial evaluation of candidate gene targets for cancer treatment prior to moving on to animal studies and clinical trials. This chapter describes a relatively simple and straightforward protocol that makes use of small interfering RNA to achieve knockdown of the candidate gene target and to evaluate the resultant effects on four aspects of cancer cell behavior: migration, invasion, proliferation, and adhesion.

Simultaneous separation of small interfering RNA and lipids using ion-pair reversed-phase liquid chromatography.

RNA interference offers a novel approach for the development of new therapeutics for targets that are otherwise "undruggable" using traditional modalities. The safety and efficacy of siRNA-based therapy mainly rely on lipid or polymer-based nanocarriers to overcome inherent barriers to a systemic delivery of siRNA. A multicomponent lipid nanoparticle (LNP) system is a promising delivery platform, typically consisting of a cationic lipid, phospholipid, PEG-containing short-chain lipid, and cholesterol. Characterization and chemical analysis of the LNP formulation is important to assure drug product stability, a key consideration for chemistry, manufacturing and control strategy. Here we report an ion-pair reversed phase UHPLC method capable of simultaneously separating both siRNA and functional lipids in LNPs with a minimal retention gap for two classes of biologically essential yet chemically distinct molecules. Key chromatographic parameters critical to the separation are discussed, including the structure of the ion-pair agent, stationary phase chemistry, column temperature and an organic additive. The results showed that the retention time of siRNA is tunable by using various ion-pair reagents. The retention factor of the siRNA exhibited a first order relationship with the number of carbons in the alkyl chain of the ion-pair reagents. In contrast, the type of ion-pair reagent has no significant impact on the separation of phospholipids. Separations using a BEH phenyl column and dibutylammonium acetate as the ion-pair reagent showed satisfactory selectivity for a range of double-stranded siRNAs and phospholipids, key components for lipid nanoparticle formulations. Furthermore, the method was applied to the separation of an experimental LNP formulation, demonstrating good selectivity for siRNA, functional lipids and their potential degradation products.

Synthetic Oligonucleotides in SPECT/CT In Vivo Imaging: Chemical Modifications, In111 Complex Formation, Incorporation into Drug Delivery Systems.

Here in we describe a solid phase synthesis of oligonucleotides bearing unnatural moiety appropriate for complex formation with In111 as well as their deprotection, isolation, and purification. We also present methods for oligonucleotides/In111 complex formulation with single and double stranded oligonucleotides of RNA nature and give an example of preparation method for one supramolecular drug delivery system (DDS) consisting of radiolabeled siRNA and positively charged peptide.

High levels of PIWI-interacting RNAs are present in the small RNA landscape of prostate epithelium from vitamin D clinical trial specimens.

BACKGROUND: Vitamin D, a hormone that acts through the nuclear vitamin D receptor (VDR), upregulates antitumorigenic microRNA in prostate epithelium. This may contribute to the lower levels of aggressive prostate cancer (PCa) observed in patients with high serum vitamin D. The small noncoding RNA (ncRNA) landscape includes many other RNA species that remain uncharacterized in prostate epithelium and their potential regulation by vitamin D is unknown. METHODS: Laser capture microdissection (LCM) followed by small-RNA sequencing was used to identify ncRNAs in the prostate epithelium of tissues from a vitamin D-supplementation trial. VDR chromatin immunoprecipitation-sequencing was performed to identify vitamin D genomic targets in primary prostate epithelial cells. RESULTS: Isolation of epithelium by LCM increased sample homogeneity and captured more diversity in ncRNA species compared with publicly available small-RNA sequencing data from benign whole prostate. An abundance of PIWI-interacting RNAs (piRNAs) was detected in normal prostate epithelium. The obligate binding partners of piRNAs, PIWI-like (PIWIL) proteins, were also detected in prostate epithelium. High prostatic vitamin D levels were associated with increased expression of piRNAs. VDR binding sites were located near several ncRNA biogenesis genes and genes regulating translation and differentiation. CONCLUSIONS: Benign prostate epithelium expresses both piRNA and PIWIL proteins, suggesting that these small ncRNA may serve an unknown function in the prostate. Vitamin D may increase the expression of prostatic piRNAs. VDR binding sites in primary prostate epithelial cells are consistent with its reported antitumorigenic functions and a role in ncRNA biogenesis.

Quantification of siRNA-Antibody Conjugates in Biological Matrices by Triplex-Forming Oligonucleotide ELISA.

The potential repertoire of short interfering RNA (siRNA) therapeutics is expanding as targeting strategies evolve. One approach to enable organ-specific delivery has been to directly conjugate siRNA to a monoclonal antibody (siRNA-mAb), analogous to antibody-drug conjugates. Detection of intact siRNA-mAb conjugates presents a bioanalytical challenge given that certain synthetic nucleotide chemical modifications and low-temperature requirements render common oligonucleotide detection assays, such as reverse transcription-polymerase chain reaction, incompatible with the immunoassay component. To circumvent these issues, we developed a triplex-forming oligonucleotide ELISA using locked nucleic acid (LNA) containing oligonucleotide probes. We demonstrate that the incorporation of these LNAs allow for an enrichment and immobilization of siRNA directly conjugated to an antibody at nondenaturing temperatures. Without further requirement for extraction or amplification, we can sensitively and specifically detect intact siRNA-mAb conjugates in complex matrices such as serum and tissue homogenate.

The potential use of Piwi-interacting RNA biomarkers in forensic body fluid identification: A proof-of-principle study.

In the forensic community, RNA profiling has been investigated as a potential method to identify body fluids. Several RNA molecules, including messenger RNA (mRNA), microRNA (miRNA) and circular RNA (circRNA), have been explored as biomarkers to distinguish different body fluids and have led to considerable interest in the development of RNA biomarkers for forensic purposes. Piwi-interacting RNA (piRNA), a class of noncoding RNAs, is a potential biomarker for body fluid identification because of its short length (˜24-32 nt) and specific expression pattern in human tissues. In this proof-of-principle study, we examined the expression levels of four carefully selected piRNAs in forensically relevant biological fluids (venous blood, saliva, semen, menstrual blood and vaginal secretions) using TaqMan quantitative real-time polymerase chain reaction (TaqMan qPCR). piR-55521, which was not detectable in saliva, can differentiate semen from other body fluids because it was strongly expressed in semen compared to the remaining three fluids (> 4000-fold change). Furthermore, piR-55521 could be detected in semen samples made from as little as 200 pg of total RNA, and addition of female component had no effect on the detection limit. Furthermore, the expression differences of other piRNAs, piR-61648, piR-43994 and piR-33151, were statistically significant between at least two types of body fluids. Stability tests also indicated that these piRNAs could be effectively detected in dried samples under laboratory and outdoor conditions for at least six months. Although limited to four piRNAs, this study suggests that the expression pattern of piRNAs could be used to identify body fluids, and that piRNA (piR-55521) is specifically expressed in semen. Such findings suggest that additional work could identify other piRNAs that could serve as biomarkers to identify body fluids.

Piwi-interacting RNA (piRNA) expression patterns in pearl oyster (Pinctada fucata) somatic tissues.

Piwi-interacting RNAs (piRNAs) belong to a recently discovered class of small non-coding RNAs whose best-understood function is repressing transposable element activity. Most piRNA studies have been conducted on model organisms and little is known about piRNA expression and function in mollusks. We performed high-throughput sequencing of small RNAs extracted from the mantle, adductor muscle, gill, and ovary tissues of the pearl oyster, Pinctada fucata. RNA species with sequences of approximately 30 nt were widely expressed in all tissues. Uridine at the 5' terminal and protection from ß-elimination at the 3' terminal suggested that these were putative piRNAs. A total of 18.0 million putative piRNAs were assigned to 2.8 million unique piRNAs, and 35,848 piRNA clusters were identified. Mapping to the reference genome showed that 25% of the unique piRNAs mapped to multiple tandem loci on the scaffold. Expression patterns of the piRNA clusters were similar within the somatic tissues, but differed significantly between the somatic and gonadal tissues. These findings suggest that in pearl oysters piRNAs have important and novel functions beyond those in the germ line.

Fast-Forward Identification of Highly Effective Artificial Small RNAs Against Different Tomato spotted wilt virus Isolates.

Artificial small RNAs (sRNAs), including artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are used to silence viral RNAs and confer antiviral resistance in plants. Here, the combined use of recent high-throughput methods for generating artificial sRNA constructs and the Tomato spotted wilt virus (TSWV)-Nicotiana benthamiana pathosystem allowed for the simple and rapid identification of amiRNAs with high anti-TSWV activity. A comparative analysis between the most effective amiRNA construct and a syn-tasiRNA construct including the four most effective amiRNA sequences showed that both were highly effective against two different TSWV isolates. These results highlight the usefulness of this high-throughput methodology for the fast-forward identification of artificial sRNAs with high antiviral activity prior to time-consuming generation of stably transformed plants.

sRNA-FISH: versatile fluorescent in situ detection of small RNAs in plants.

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA-FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co-localization of miR2275 and a 24-nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi-photon fluorescence excitation microscopy can be used to separate the target sRNA-FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA-FISH signals can be imaged using super-resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super-resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step-by-step sRNA-FISH protocol for studying sRNAs at the cellular and even subcellular level.

BVDV Npro protein mediates the BVDV induced immunosuppression through interaction with cellular S100A9 protein.

The innate immune response is a vital part of the body's antiviral defense system. The innate immune response is initiated by various receptor interactions, including danger associated molecular patterns (DAMPs). The S100A9 is a member of the DAMPs protein family and, is released by activated phagocytic cells such as neutrophils, monocytes, macrophages or endothelial cells, and S100A9 induces its effect through TLR4/MyD88 pathway. Bovine viral diarrhea virus (BVDV) is one of the major devastating disease in the cattle industry worldwide. It shows its effect through immunosuppression and develops persistent infection in calves born from infected cows. The current study revealed that BVDV potentially induced immunosuppression by the interaction of BVDV Npro protein with cellular S100A9 protein. The Inhibition of S100A9 protein expression by small interfering RNA (siRNA) enhanced the virus replication in infected cells. Overexpression of bovine S100A9 enhanced the ncpBVDV2a 1373 mediated Type-I interferon production. A co-immunoprecipitation experiment demonstrated a strong interaction between ncp BVDV2a 1373 Npro protein and cellular S100A9 protein. This suggested that BVDV Npro reduced the S100A9 protein availability/activity in infected cells, resulting in reduced Type-I interferon production. A further study of S100A9-BVDV interaction will be need for better understanding of BVDV pathophysiology.

Improved Boronate Affinity Electrophoresis by Optimization of the Running Buffer for a Single-step Separation of piRNA from Mouse Testis Total RNA.

Here we examined optimization of the running buffer in boronate affinity electrophoresis for improved separation of PIWI-interacting RNA (piRNA) with 2'-O-methylated ribose in 3'-terminal nucleotide. The use of Good's buffer, such as HEPES, significantly increased the separation efficiency for piRNA over normal RNA with free 3'-terminal ribose, and retained an ability to resolve the difference by at least 4-nucleotide lengths in the target piRNAs. We also demonstrated a single-step separation of piRNA from mouse testis total RNA.

A fast and efficient protocol for small RNA extraction in Japanese plum and other Prunus species

Background: Small ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum (Prunus domestica) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV-Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry. Results: We describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPV­P. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159. Conclusion: We propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species.