Multitarget Stool RNA Test for Colorectal Cancer Screening.

Importance: Noninvasive tests for colorectal cancer screening must include sensitive detection of colorectal cancer and precancerous lesions. These tests must be validated for the intended-use population, which includes average-risk individuals 45 years or older. Objective: To evaluate the sensitivity and specificity of a noninvasive, multitarget stool RNA (mt-sRNA) test (ColoSense) test compared with results from a colonoscopy. Design, Setting, and Participants: This phase 3 clinical trial (CRC-PREVENT) was a blinded, prospective, cross-sectional study to support a premarket approval application for a class III medical device. A total of 8920 participants were identified online using social media platforms and enrolled from June 2021 to June 2022 using a decentralized nurse call center. All participants completed the mt-sRNA test, which incorporated a commercially available fecal immunochemical test (FIT), concentration of 8 RNA transcripts, and participant-reported smoking status. Stool samples were collected prior to participants completing a colonoscopy at their local endoscopy center. The mt-sRNA test results (positive or negative) were compared with index lesions observed on colonoscopy. Over the course of 12 months, individuals 45 years and older were enrolled in the clinical trial using the decentralized recruitment strategy. Participants were enrolled from 49 US states and obtained colonoscopies at more than 3800 different endoscopy centers. Main Outcomes and Measures: The primary outcomes included the sensitivity of the mt-sRNA test for detecting colorectal cancer and advanced adenomas and the specificity for no lesions on colonoscopy. Results: The mean (range) age of participants was 55 (45-90) years, with 4% self-identified as Asian, 11% as Black, and 7% as Hispanic. Of the 8920 eligible participants, 36 (0.40%) had colorectal cancer and 606 (6.8%) had advanced adenomas. The mt-sRNA test sensitivity for detecting colorectal cancer was 94%, sensitivity for detecting advanced adenomas was 46%, and specificity for no lesions on colonoscopy was 88%. The mt-sRNA test showed significant improvement in sensitivity for colorectal cancer (94% vs 78%; McNemar P = .01) and advanced adenomas (46% vs 29%; McNemar P < .001) compared with results of the FIT. Conclusions and Relevance: In individuals 45 years and older, the mt-sRNA test showed high sensitivity for colorectal neoplasia (colorectal cancer and advanced adenoma) with significant improvement in sensitivity relative to the FIT. Specificity for no lesions on colonoscopy was comparable to existing molecular diagnostic tests. Trial Registration: ClinicalTrials.gov Identifier: NCT04739722.

Quantitative and Qualitative Assessment of Small RNA Preparations.

Recent advances in high-throughput sequencing have shed new light on the diversity of small noncoding RNA (sncRNA) classes and their crucial roles in gene regulation and disease. One key step in sncRNA profiling consists in their quantification and assessment of their degradation extent. In this chapter, we will describe different gold standard methods used to achieve both purposes before using the sncRNAs in downstream applications.

Fluorescence In Situ Hybridization of Small Non-Coding RNAs.

The determination of the cellular localization of a noncoding RNA (ncRNA) is highly helpful to decipher its function. RNA-FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA-FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA-FISH protocol that we developed to study the expression and localization of satellite III (SATIII) RNAs. This specific class of ncRNAs is expressed in response to various cellular stresses, including heat shock. The protocol is based on the use of a biotinylated LNA probe subsequently detected by a Streptavidin, Alexa Fluor® 488 conjugate. A protocol allowing efficient coupling of RNA-FISH and protein detection by immunofluorescence is also described as well as the bioinformatics pipeline, Substructure Analyzer, we recently developed to automate fluorescence signal analysis.

Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in E. coli.

Almost two-thirds of the microbiome's biomass has been predicted to be in a non-proliferating, and thus dormant, growth state. It is assumed that dormancy goes hand in hand with global downregulation of gene expression. However, it remains largely unknown how bacteria manage to establish this resting phenotype at the molecular level. Recently small non-protein-coding RNAs (sRNAs or ncRNAs) have been suggested to be involved in establishing the non-proliferating state in bacteria. Here, we have deep sequenced the small transcriptome of Escherichia coli in the exponential and stationary phases and analyzed the resulting reads by a novel biocomputational pipeline STARPA (Stable RNA Processing Product Analyzer). Our analysis reveals over 12,000 small transcripts enriched during both growth stages. Differential expression analysis reveals distinct sRNAs enriched in the stationary phase that originate from various genomic regions, including transfer RNA (tRNA) fragments. Furthermore, expression profiling by Northern blot and RT-qPCR analyses confirms the growth phase-dependent expression of several enriched sRNAs. Our study adds to the existing repertoire of bacterial sRNAs and suggests a role for some of these small molecules in establishing and maintaining stationary phase as well as the bacterial stress response. Functional characterization of these detected sRNAs has the potential of unraveling novel regulatory networks central for stationary phase biology.

A mouse tissue atlas of small noncoding RNA.

Small noncoding RNAs (ncRNAs) play a vital role in a broad range of biological processes both in health and disease. A comprehensive quantitative reference of small ncRNA expression would significantly advance our understanding of ncRNA roles in shaping tissue functions. Here, we systematically profiled the levels of five ncRNA classes (microRNA [miRNA], small nucleolar RNA [snoRNA], small nuclear RNA [snRNA], small Cajal body-specific RNA [scaRNA], and transfer RNA [tRNA] fragments) across 11 mouse tissues by deep sequencing. Using 14 biological replicates spanning both sexes, we identified that ∼30% of small ncRNAs are distributed across the body in a tissue-specific manner with some also being sexually dimorphic. We found that some miRNAs are subject to "arm switching" between healthy tissues and that tRNA fragments are retained within tissues in both a gene- and a tissue-specific manner. Out of 11 profiled tissues, we confirmed that brain contains the largest number of unique small ncRNA transcripts, some of which were previously annotated while others are identified in this study. Furthermore, by combining these findings with single-cell chromatin accessibility (scATAC-seq) data, we were able to connect identified brain-specific ncRNAs with their cell types of origin. These results yield the most comprehensive characterization of specific and ubiquitous small RNAs in individual murine tissues to date, and we expect that these data will be a resource for the further identification of ncRNAs involved in tissue function in health and dysfunction in disease.

Small RNA modifications in Alzheimer's disease.

Background While significant advances have been made in uncovering the aetiology of Alzheimer's disease and related dementias at the genetic level, molecular events at the epigenetic level remain largely undefined. Emerging evidence indicates that small non-coding RNAs (sncRNAs) and their associated RNA modifications are important regulators of complex physiological and pathological processes, including aging, stress responses, and epigenetic inheritance. However, whether small RNAs and their modifications are altered in dementia is not known. Methods We performed LC-MS/MS-based, high-throughput assays of small RNA modifications in post-mortem samples of the prefrontal lobe cortices of Alzheimer's disease (AD) and control individuals. We noted that some of the AD patients has co-occurring vascular cognitive impairment-related pathology (VaD). Findings We report altered small RNA modifications in AD samples compared with normal controls. The 15-25-nucleotide (nt) RNA fraction of these samples was enriched for microRNAs, whereas the 30-40-nt RNA fraction was enriched for tRNA-derived small RNAs (tsRNAs), rRNA-derived small RNAs (rsRNAs), and YRNA-derived small RNAs (ysRNAs). Interestingly, most of these altered RNA modifications were detected both in the AD and AD with co-occurring vascular dementia subjects. In addition, sequencing of small RNA in the 30-40-nt fraction from AD cortices revealed reductions in rsRNA-5S, tsRNA-Tyr, and tsRNA-Arg. Interpretation These data suggest that sncRNAs and their associated modifications are novel signals that may be linked to the pathogenesis and development of Alzheimer's disease. Fund NIH grants (R01HL122770, R01HL091905, 1P20GM130459, R01HD092431, P50HD098593, GM103440), AHA grant (17IRG33370128), Sigmund Gestetner Foundation Fellowship to P Kehoe.

Comparability of the small RNA secretome across human biofluids concomitantly collected from healthy adults.

Small extracellular vesicles (sEV) are nano-sized (40-150 nm), membrane-encapsulated vesicles that are released by essentially all cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA) and other small non-coding RNA (ncRNA), that can alter the phenotype of recipient cells. sEV are present in essentially all extracellular biofluids, including serum, urine and saliva, and offer a new avenue for discovery and development of novel biomarkers of various disease states and exposures. The objective of this study was to systematically interrogate similarities and differences between sEV ncRNA derived from saliva, serum and urine, as well as cell-free small ncRNA (cf-ncRNA) from serum. Saliva, urine and serum were concomitantly collected from 4 healthy donors to mitigate potential bias that can stem from interpersonal and temporal variability. sEV were isolated from each respective biofluid, along with cf-RNA from serum. sEV were isolated from the respective biofluids via differential ultracentrifugation with a 30% sucrose cushion to minimize protein contamination. Small RNA-sequencing was performed on each sample, and cluster analysis was performed based on ncRNA profiles. While some similarities existed in terms of sEV ncRNA cargo across biofluids, there are also notable differences in ncRNA class and ncRNA secretion, with sEV in each biofluid bearing a unique ncRNA profile, including major differences in composition by ncRNA class. We conclude that sEV ncRNA cargo varies according to biofluid, so thus should be carefully selected and interpreted when designing or contrasting translational or epidemiological studies.

Functional analyses of mammalian virus 5'UTR-derived, small RNAs that regulate virus translation.

Enterovirus A71 (EV-A711) RNA contains an internal ribosomal entry site (IRES) to direct cap-independent translation. IRES-dependent translation requires the host's translation initiation factors and IRES-associated trans-acting factors (ITAFs). We previously showed that hnRNP A1, the mRNA stability factor HuR, and the RISC subunit Argonaute 2 (Ago2) are ITAFs that associate with stem loop II (SL-II) of the IRES and promote IRES-dependent translation. By contrast, the mRNA decay factor AUF1 is a negative-acting ITAF that also binds SL-II. Moreover, the small RNA-processing enzyme Dicer produces at least four virus-derived, small RNAs (vsRNAs 1-4) from the EV-A71 5'UTR in infected cells. One of these, vsRNA1, derived from SL-II, inhibits IRES activity via an unknown mechanism. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with SL-II. This presents a possible mechanism by which vsRNA1 could control association of ITAFs with the IRES and modulate viral translation. Here, we describe methods for functional analyses of vsRNA1-mediated regulation of IRES activity. These methods should be applicable to other virus-derived, small RNAs as well.

Small RNA In Situ Hybridizations on Sections of Arabidopsis Embryos.

Small RNAs mediate posttranscriptional gene silencing in plants and animals. This often occurs in specific cell or tissue types and can be necessary for their differentiation. Determining small RNA (sRNA) localization patterns at cellular resolution can therefore provide information on the corresponding gene regulatory processes they are involved in. Recent improvements with in situ hybridization methods have allowed them to be applied to sRNAs. Here we describe an in situ hybridization protocol to detect sRNAs from sections of early staged Arabidopsis thaliana (Arabidopsis) embryos.

RNU6B, a frequent reference in miRNA expression studies, differentiates between deaths caused by hypothermia and chronic cardiac ischemia.

Here, we tested the usefulness of small non-coding RNAs as references in quantitative RT-PCR expression analyses in hypothermia and chronic cardiac ischemia as the primary causes of death. Cq values of RNU6B, SCARNA17, SNORD25, and SNORA73A were determined from human cadaver samples of hypothermia and cardiac deaths. Average Cq values of RNU6B were higher in hypothermic and average SCARNA17 Cq values in chronic ischemic samples, but no difference in SNORD25 and SNORA73A Cq values could be seen between the groups. RNU6B expression levels were calculated using SNORD25, SNORA73A, and their combination as the reference in normalization. Expression of RNU6B, a widely used reference, was found to be significantly lower in hypothermia than in chronic cardiac ischemia. In these conditions, RNU6B is a useful marker differentiating hypothermia deaths from chronic ischemic heart disease deaths, but not a valid reference for normalization in expression studies.

Isolation of enriched small RNA from cell-lysate using on-chip isotachophoresis.

In spite of the growing interest in the roles and applications of small RNAs (sRNAs), sRNA isolation methods are inconsistent, tedious, and dependent on the starting number of cells. In this work, we employ ITP to isolate sRNAs from the cell-lysate of K562 (chronic myelogenous leukemia) cells in a polydimethylsiloxane (PDMS) mesofluidic device. Our method specifically purifies sRNA of <60 nucleotides from lysate of a wide range of cell number spanning from 100 to 1 000 000 cells. We measured the amount of sRNA using the Agilent Bioanalyzer and further verified the extraction efficiency by reverse transcription quantitative PCR. Our method was shown to be more efficient in sRNA extraction than commercial sRNA isolation kits, especially when using smaller numbers of starting cells. Our assay presents a simple and rapid sRNA extraction method with 20 min assay time and no intermediate transfer steps.

Cell-Free, Embryo-Specific sncRNA as a Molecular Biological Bridge between Patient Fertility and IVF Efficiency.

Small noncoding RNAs (sncRNAs) are key regulators of the majority of human reproduction events. Understanding their function in the context of gametogenesis and embryogenesis will allow insight into the possible causes of in vitro fertilization (IVF) implantation failure. The aim of this study was to analyze the sncRNA expression profile of the spent culture media on day 4 after fertilization and to reveal a relationship with the morphofunctional characteristics of gametes and resultant embryos, in particular, with the embryo development and implantation potential. Thereto, cell-free, embryo-specific sncRNAs were identified by next generation sequencing (NGS) and quantified by reverse transcription coupled with polymerase chain reaction (RT-PCR) in real-time. Significant differences in the expression level of let-7b-5p, let-7i-5p, piR020401, piR16735, piR19675, piR20326, and piR17716 were revealed between embryo groups of various morphological gradings. Statistically significant correlations were found between the expression profiles of piR16735 and piR020401 with the oocyte-cumulus complex number, let-7b-5p and piR020401 with metaphase II oocyte and two pronuclei embryo numbers, let-7i-5p and piR20497 with the spermatozoid count per milliliter of ejaculate, piR19675 with the percentage of linearly motile spermatozoids, let-7b-5p with the embryo development grade, and let-7i-5p with embryo implantation. According to partial least squares discriminant analysis (PLS-DA), the expression levels of let-7i-5p (Variable Importance in Projection score (VIP) = 1.6262), piR020401 (VIP = 1.45281), and piR20497 (VIP = 1.42765) have the strongest influences on the implantation outcome.

Detection, infection dynamics and small RNA response against Culex Y virus in mosquito-derived cells.

Many insect cell lines are persistently infected with insect-specific viruses (ISV) often unrecognized by the scientific community. Considering recent findings showing the possibility of interference between arbovirus and ISV infections, it is important to pay attention to ISV-infected cell lines. One example is the Entomobirnavirus, Culex Y virus (CYV). Here we describe the detection of CYV using a combination of small RNA sequencing, electron microscopy and PCR in mosquito cell lines Aag2, U4.4 and C7-10. We found CYV-specific small RNAs in all three cell lines. Interestingly, the magnitude of the detected viral RNA genome is variable among cell passages and leads to irregular detection via electron microscopy. Gaining insights into the presence of persistent ISV infection in commonly used mosquito cells and their interactions with the host immune system is beneficial for evaluating the outcome of co-infections with arboviruses of public health concern.

tRNA-halves are prognostic biomarkers for patients with prostate cancer.

OBJECTIVE: Noncoding RNAs play an important role in carcinogenesis; a number of tRNA-halves are expressed in response to androgen stimulation and are involved in prostate cancer (CaP) initiation and progression. In this study, we evaluated the expression profile of androgen-dependent tRNA-halves in CaP. MATERIALS AND METHODS: Total RNA was isolated from formalin-fixed paraffin-embedded 58 CaP, and 25 benign prostate hyperplasia tissues. We also studied the expression in serum from 49 localized and 21 metastatic castration-resistant CaP samples. The ligation of a RNA adaptor molecule to the tRNA-halves allowed the specific amplification of 3'/5'-tRNA-halves using quantitative TaqMan reverse transcription polymerase chain reaction. The expression levels were correlated with clinicopathological parameters as well as prostate-specific antigen recurrence free survival. RESULTS: 5'-tRNA-Asp-GUC-half and 3'-tRNA-Asp-GUC-half were up-regulated in CaP tissues compared with benign prostate hyperplasia tissues. An increased expression level of all the 5 candidate tRNA-halves was associated with adverse clinicopathological parameters (pT-stage, pN-stage, prostate-specific antigen, International Society of Urological Pathology /ISUP grade) and a shorter time to biochemical relapse. In serum, 5'-tRNA-Glu-CUC-half was circulating at a higher level in metastatic castration-resistant CaP patients compared to patients with localized CaP. CONCLUSIONS: The androgen-dependent tRNA-halves can potentially act as biomarkers to monitor and predict the progression of CaP.

The Stringent Response-Regulated sRNA Transcriptome of Borrelia burgdorferi.

The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by RelBbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that RelBbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were RelBbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5' untranslated region sRNAs. RelBbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.

High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions.

We are just beginning to unravel the myriad of interactions in which non-coding RNAs participate. The intricate RNA interactome is the foundation of many biological processes, including bacterial virulence and human disease, and represents unexploited resources for the development of potential therapeutic interventions. However, identifying specific associations of a given RNA from the multitude of possible binding partners within the cell requires robust high-throughput systems for their rapid screening. Here, we present the first demonstration of functional-RNA arrays as a novel platform technology designed for the study of such interactions using immobilized, active RNAs. We have generated high-density RNA arrays by an innovative method involving surface-capture of in vitro transcribed RNAs. This approach has significant advantages over existing technologies, particularly in its versatility in regards to binding partner character. Indeed, proof-of-principle application of RNA arrays to both RNA-small molecule and RNA-RNA pairings is demonstrated, highlighting their potential as a platform technology for mapping RNA-based networks and for pharmaceutical screening. Furthermore, the simplicity of the method supports greater user-accessibility over currently available technologies. We anticipate that functional-RNA arrays will find broad utility in the expanding field of RNA characterization.

Detection of mycobacterial small RNA in the bacterial culture supernatant and plasma of patients with active tuberculosis.

Bacterial small RNA (sRNA) has been shown to play an important role in control of bacteria virulence, stress response and physiological metabolism by post-transcriptional regulation of gene expression. However, there were few reports about bacterial sRNA as a biomarker of infection. To test the potential role of sRNA in indicating infection of Mycobacterium tuberculosis, total RNA were extracted from the filtrated bacterial cultural supernatant. After synthesis of cDNA by reverse transcription, four Mycobacterial sRNAs including ASdes, ASpks, AS1726, and AS1890, which have been experimentally confirmed by Kristine B in the year of 2009, were detected by real time PCR. The specificity was verified by sequencing of the amplified products. Moreover, we demonstrate that the presence of sRNA Asdes in plasma of 55.56% (15/27) TB patients and 25.00% (6/24) normal controls with BCG vaccination (P < 0.05). Our results suggest that bacterial non-coding sRNA can be detected from either bacterial culture supernatants or patient's plasma. Detecting of Mycobacterial sRNA provides a rapid and relatively noninvasive approach for diagnosing disease and could be developed as a biomarker to identify patients with active tuberculosis to help make informed decisions about proper therapies.1.

High-throughput sequencing identification and characterization of potentially adhesion-related small RNAs in Streptococcus mutans.

PURPOSE: Adherence capacity is one of the principal virulence factors of Streptococcus mutans, and adhesion virulence factors are controlled by small RNAs (sRNAs) at the post-transcriptional level in various bacteria. Here, we aimed to identify and decipher putative adhesion-related sRNAs in clinical strains of S. mutans. METHODOLOGY: RNA deep-sequencing was performed to identify potential sRNAs under different adhesion conditions. The expression of sRNAs was analysed by quantitative real-time PCR (qRT-PCR), and bioinformatic methods were used to predict the functional characteristics of sRNAs. RESULTS: A total of 736 differentially expressed candidate sRNAs were predicted, and these included 352 sRNAs located on the antisense to mRNA (AM) and 384 sRNAs in intergenic regions (IGRs). The top 7 differentially expressed sRNAs were successfully validated by qRT-PCR in UA159, and 2 of these were further confirmed in 100 clinical isolates. Moreover, the sequences of two sRNAs were conserved in other Streptococcus species, indicating a conserved role in such closely related species. A good correlation between the expression of sRNAs and the adhesion of 100 clinical strains was observed, which, combined with GO and KEGG, provides a perspective for the comprehension of sRNA function annotation. CONCLUSION: This study revealed a multitude of novel putative adhesion-related sRNAs in S. mutans and contributed to a better understanding of information concerning the transcriptional regulation of adhesion in S. mutans.

Microfluidics-based digital quantitative PCR for single-cell small RNA quantification.

Quantitative analyses of small RNAs at the single-cell level have been challenging because of limited sensitivity and specificity of conventional real-time quantitative PCR methods. A digital quantitative PCR (dqPCR) method for miRNA quantification has been developed, but it requires the use of proprietary stem-loop primers and only applies to miRNA quantification. Here, we report a microfluidics-based dqPCR (mdqPCR) method, which takes advantage of the Fluidigm BioMark HD system for both template partition and the subsequent high-throughput dqPCR. Our mdqPCR method demonstrated excellent sensitivity and reproducibility suitable for quantitative analyses of not only miRNAs but also all other small RNA species at the single-cell level. Using this method, we discovered that each sperm has a unique miRNA profile.