RNA In Situ Hybridization on Plant Tissue Sections: Expression Analysis at Cellular Resolution.

RNA in situ hybridization offers a means to study the spatial expression of candidate genes by making use of specific, labelled RNA probes on thin tissue sections. Unlike other methods, such as promoter GUS fusions, for which all regulatory sequences should be available and transgenic plants have to be generated, RNA in situ hybridization allows specific and direct detection of even low abundant transcripts at cellular resolution. Although various protocols exist, the results published throughout the literature indicate a very obvious problem of the technique: each step has the potential to affect the outcome, that is, the signal strength, presence or absence of background, and visibility of individual cells. The protocol described here tries to avoid all these problems by addressing each step in detail and providing advice regarding critical steps for a distinct visualization of gene expression on intact tissue sections without any background.

Integration of the Ligase Chain Reaction with the CRISPR-Cas12a System for Homogeneous, Ultrasensitive, and Visual Detection of microRNA.

The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis.

Optimized protocol for single-molecule RNA FISH to visualize gene expression in S. cerevisiae.

Single-molecule RNA fluorescence in situ hybridization (smFISH) allows subcellular visualization, localization, and quantification of endogenous RNA molecules in fixed cells. The spatial and intensity information of each RNA can be used to distinguish mature from nascent transcripts inside each cell, revealing both past and instantaneous transcriptional activity. Here, we describe an optimized protocol for smFISH in Saccharomyces cerevisiae with optimized lyticase digestion time and hybrization steps for more homogenous results. For complete details on the use and execution of this protocol, please refer to Donovan et al. (2019).

Development of a Broadly Applicable Cas12a-Linked Beam Unlocking Reaction for Sensitive and Specific Detection of Respiratory Pathogens Including SARS-CoV-2.

The outbreak of novel coronavirus SARS-CoV-2 has caused a worldwide threat to public health. COVID-19 patients with SARS-CoV-2 infection can develop clinical symptoms that are often confused with the infections of other respiratory pathogens. Sensitive and specific detection of SARS-CoV-2 with the ability to discriminate from other viruses is urgently needed for COVID-19 diagnosis. Herein, we streamlined a highly efficient CRISPR-Cas12a-based nucleic acid detection platform, termed Cas12a-linked beam unlocking reaction (CALIBURN). We show that CALIBURN could detect SARS-CoV-2 and other coronaviruses and influenza viruses with little cross-reactivity. Importantly, CALIBURN allowed accurate diagnosis of clinical samples with extremely low viral loads, which is a major obstacle for the clinical applications of existing CRISPR diagnostic platforms. When tested on the specimens from SARS-CoV-2-positive and negative donors, CALIBURN exhibited 73.0% positive and 19.0% presumptive positive rates and 100% specificity. Moreover, unlike existing CRISPR detection methods that were mainly restricted to respiratory specimens, CALIBURN displayed consistent performance across both respiratory and nonrespiratory specimens, suggesting its broad specimen compatibility. Finally, using a mouse model of SARS-CoV-2 infection, we demonstrated that CALIBURN allowed detection of coexisting pathogens without cross-reactivity from a single tissue specimen. Our results suggest that CALIBURN can serve as a versatile platform for the diagnosis of COVID-19 and other respiratory infectious diseases.

RASP: an atlas of transcriptome-wide RNA secondary structure probing data.

RNA molecules fold into complex structures that are important across many biological processes. Recent technological developments have enabled transcriptome-wide probing of RNA secondary structure using nucleases and chemical modifiers. These approaches have been widely applied to capture RNA secondary structure in many studies, but gathering and presenting such data from very different technologies in a comprehensive and accessible way has been challenging. Existing RNA structure probing databases usually focus on low-throughput or very specific datasets. Here, we present a comprehensive RNA structure probing database called RASP (RNA Atlas of Structure Probing) by collecting 161 deduplicated transcriptome-wide RNA secondary structure probing datasets from 38 papers. RASP covers 18 species across animals, plants, bacteria, fungi, and also viruses, and categorizes 18 experimental methods including DMS-seq, SHAPE-Seq, SHAPE-MaP, and icSHAPE, etc. Specially, RASP curates the up-to-date datasets of several RNA secondary structure probing studies for the RNA genome of SARS-CoV-2, the RNA virus that caused the on-going COVID-19 pandemic. RASP also provides a user-friendly interface to query, browse, and visualize RNA structure profiles, offering a shortcut to accessing RNA secondary structures grounded in experimental data. The database is freely available at http://rasp.zhanglab.net.

Comparison of Primer-Probe Sets among Different Master Mixes for Laboratory Screening of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

BACKGROUND: There is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and a surge of SARS-CoV-2 cases, especially in limited-resource settings. Therefore, the combination of an optimal assay kit is necessary. METHODS: We compared the ability to screen SARS-CoV-2 among three primer-probe sets in two different master mixes, Invitrogen™ SuperScript™ III One-Step RT-PCR and LightCycler Multiplex RNA Virus Master. RESULTS: The assay with TIB-Molbiol, IDT, and Phu Sa sets for LightCycler Multiplex RNA Virus Master or Invitrogen™ SuperScript™ III One-Step RT-PCR showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. R squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. CONCLUSIONS: Our findings indicated that TIB-Molbiol, IDT, and Phu Sa primer-probe sets could be beneficial for the laboratory screening of SARS-CoV-2 by RT-qPCR assay of E gene. There is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for COVID-19.

Oligonucleotide Probe for Transcriptome in Vivo Analysis (TIVA) of Single Neurons with Minimal Background.

Messenger RNA (mRNA) isolated from single cells can generate powerful biological insights, including the discovery of new cell types with unique functions as well as markers potentially predicting a cell's response to various therapeutic agents. We previously introduced an oligonucleotide-based technique for site-selective, photoinduced biotinylation and capture of mRNA within a living cell called transcriptome in vivo analysis (TIVA). Successful application of the TIVA technique hinges upon its oligonucleotide probe remaining completely inert (or "caged") to mRNA unless photoactivated. To improve the reliability of TIVA probe caging in diverse and challenging biological conditions, we applied a rational design process involving iterative modifications to the oligonucleotide construct. In this work, we discuss these design motivations and present an optimized probe with minimal background binding to mRNA prior to photolysis. We assess its caging performance through multiple in vitro assays including FRET analysis, native gel comigration, and pull down with model mRNA transcripts. Finally, we demonstrate that this improved probe can also isolate mRNA from single living neurons in brain tissue slices with excellent caging control.

Primer design for quantitative real-time PCR for the emerging Coronavirus SARS-CoV-2.

In December 2019, a new coronavirus disease (COVID-19) outbreak occurred in Wuhan, China. Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), which is the seventh coronavirus known to infect humans, is highly contagious and has rapidly expanded worldwide since its discovery. Quantitative nucleic acid testing has become the gold standard for diagnosis and guiding clinical decisions regarding the use of antiviral therapy. However, the RT-qPCR assays targeting SARS-CoV-2 have a number of challenges, especially in terms of primer design. Primers are the pivotal components of a RT-qPCR assay. Once virus mutation and recombination occur, it is difficult to effectively diagnose viral infection by existing RT-qPCR primers. Some primers and probes have also been made available on the WHO website for reference. However, no previous review has systematically compared the previously reported primers and probes and described how to design new primers in the event of a new coronavirus infection. This review focuses on how primers and probes can be designed methodically and rationally, and how the sensitivity and specificity of the detection process can be improved. This brief review will be useful for the accurate diagnosis and timely treatment of the new coronavirus pneumonia.

Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription-PCR (RT-qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR analytical efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charité) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.

Analysis of Gene Expression in Fasciola hepatica Juveniles and Adults by In Situ Hybridization.

In situ hybridization (ISH) is a technique used for the spatial localization of nucleic acids within tissues and cells. It is based on the ability of labeled nucleic acids (probes) to hybridize under the right conditions with the nucleic acids present in fixed biological specimens. In this chapter, we describe protocols for detection of RNA by ISH using digoxigenin (DIG)-labeled probes for Fasciola hepatica adults (in cryosections, given their large size) and for newly excysted juveniles (NEJs, which are ideally suited given their small size for whole-mount ISH). We describe fluorogenic and chromogenic protocols, respectively, but the detection methods can be easily interchanged by using the appropriate enzyme-conjugated antibodies and detection solutions.

Development of a Taqman-based real-time PCR assay for detection of porcine sapelovirus infection in pigs.

The objective of the present study was to develop a rapid, simple, specific and sensitive Taqman-based real-time PCR assay for porcine sapelovirus (PSV) detection. Specific primers and probe were designed from the five untranslated regions (UTRs) of the viral genome. The detection limit of the real-time PCR was 102 copies. The specificity of the Taqman real-time PCR assay was evaluated using other animal viruses and nuclease free water as a negative control. Strong fluorescent signals were obtained only in the detection of PSV real-time PCR and conventional RT-PCR were preformed simultaneously on 90 faecal samples. Based on conventional RT-PCR study 17.7% (16/90) of the faecal samples were positive for PSV. Whereas 21 of 90 samples (23.3%) were positive by real-time RT-PCR. The results showed that real-time PCR was more sensitive than the conventional RT-PCR assay. In conclusion, the Taqman real-time PCR assay for detection of PSV developed, herein, is sensitive, specific, and reliable. This assay will be useful for clinical diagnosis, epidemiological, and pathogenesis studies.

5'-Monopyrene and 5'-Bispyrene 2'-O-methyl RNA Probes for Detection of RNA Mismatches.

Progress in synthesis of novel fluorescent oligonucleotides has provided effective instruments for nucleic acid detection. Pyrene conjugated oligonucleotides have demonstrated their effectiveness as fluorescent hybridization probes. Here we describe the synthesis, isolation, and analysis of 5'-monopyrene and 5'-bispyrene conjugates of oligo(2'-O-methylribonucleotides) and their application as probes for fluorescent detection of mismatches in RNA targets.

Rational design and development of a universal baby spinach-based sensing platform for the detection of biomolecules.

Here, we designed and developed a Universal Baby Spinach-based Probe (UBSP) for biomolecule detection by introducing a DNA repressor containing a target recognition element. By employing different interaction modes between targets and repressors, we applied the UBSP to detect diverse classes of analytes, including microRNA, proteins, and heavy metal ions.

Cyclodextrin supramolecular inclusion-enhanced pyrene excimer switching for highly selective detection of RNase H.

Here, we report a novel fluorescence method for the highly selective and sensitive detection of RNase H by combining the use of a dual-pyrene-labeled DNA/RNA duplex with supramolecular inclusion-enhanced fluorescence. Initially, the probe is in the "off" state due to the rigidness of the double-stranded duplex, which separates the two pyrene units. In the presence of RNase H, the RNA strand of the DNA/RNA duplex will be hydrolyzed, and the DNA strand transforms into a hairpin structure, bringing close the two pyrene units which in turn enter the hydrophobic cavity of a γ-cyclodextrin. As a result, the pyrene excimer emission is greatly enhanced, thereby realizing the detection of RNase H activity. Under optimal conditions, RNase H detection can be achieved in the range from 0.08 to 4 U/mL, with a detection limit of 0.02 U/mL.

CIRDES: an efficient genome-wide method for in vivo RNA-RNA interactome analysis.

Complex RNA-RNA interactions underlie fundamental biological processes. However, a large number of RNA-RNA interactions remain unknown. Most existing methods used to map RNA-RNA interactions are based on proximity ligation, but these strategies also capture a huge amount of intramolecular RNA secondary structures, making it almost impossible to detect most RNA-RNA interactions. To overcome this limitation, we developed an efficient, genome-wide method, Capture Interacting RNA and Deep Sequencing (CIRDES) for in vivo capturing of the RNA interactome. We designed multiple 20-nt CIRDES probes tiling the whole RNA sequence of interest. This strategy obtained high selectivity and low background noise proved by qRT-PCR data. CIRDES enriched target RNA and its interacting RNAs from cells crosslinked by formaldehyde in high efficiency. After hybridization and purification, the captured RNAs were converted to the cDNA library after a highly efficient ligation to a 3' end infrared-dye-conjugated RNA adapter based on adapter ligation library construction. Using CIRDES, we detected highly abundant known interacting RNA, as well as a large number of novel targets of U6 snRNA. The enrichment of U4 snRNA, which interacts with U6, confirmed the robustness of the identification of the RNA-RNA interaction by CIRDES. These results suggest that the CIRDES is an efficient strategy for genome-wide RNA-RNA interactome analysis.

RNA Stable Isotope Probing (RNA-SIP).

Stable isotope probing is a combined molecular and isotopic technique used to probe the identity and function of uncultivated microorganisms within environmental samples. Employing stable isotopes of common elements such as carbon and nitrogen, RNA-SIP exploits an increase in the buoyant density of RNA caused by the active metabolism and incorporation of heavier mass isotopes into the RNA after cellular utilization of labeled substrates pulsed into the community. Labeled RNAs are subsequently separated from unlabeled RNAs by density gradient centrifugation followed by identification of the RNAs by sequencing. Therefore, RNA stable isotope probing is a culture-independent technique that provides simultaneous information about microbiome community, composition and function. This chapter presents the detailed protocol for performing an RNA-SIP experiment, including the formation, ultracentrifugation, and fractional analyses of stable isotope-labeled RNAs extracted from environmental samples.

In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.

A novel versatile locked nucleic acid modified molecular beacon probe (LNA-MB) was developed for imaging intracellular precursor miRNAs (pre-miRNAs) and disturbing Dicer-mediated cleavage process. The target recognition reaction between the smart probe and pre-miRNA can not only induce the conformational changes of probe and block the Dicer cleavage site, but also inhibit the cleavage process, and then achieve down-regulation of miRNA expression. Simultaneously, the target recognition reaction broke the fluorescence resonance energy transfer (FRET) between fluorophore donor FAM and acceptor TAMRA, which were labelled on the LNA-MB probe, further induced the relevant change of fluorescence signal, and then achieved imaging analysis of pre-miRNA and inhibition events in situ. Both in vitro and in single living cell studies showed that the versatile probes exhibited a remarkable performance in targeting with pre-miRNA-21, and nearly 65% downregulation of mature miRNA-21 was achieved with 100 nM probes. All investigations demonstrate that the proposed strategy represents a promising alternative for regulating and inhibiting endogenous disease-associated RNAs, then further for achieving therapeutic outcomes in personalized treatments.

In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay.

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.

Ratiometric enhanced fluorometric determination and imaging of intracellular microRNA-155 by using carbon dots, gold nanoparticles and rhodamine B for signal amplification.

An ultrasensitive and highly reliable ratiometric assay is described for the determination of microRNA-155. It works at the attomolar concentration level and has high selectivity which warrants its potential application in cancer biomarker tracking. The excellent performance of this method results from (a) the use of a hybrid conjugate prepared from Rhodamine B (RhB), carbon dots (CDs) and probe-microRNA, and (b) from the measurement of fluorescence resonance energy transfer (FRET) that is observed in the AuNP/target-microRNA system as a result of RNA hybridization. The dye RhB (emission peak at 580 nm) serves as an internal reference. The sensitivity of this assay is increased by about 30% because of the broad emissions of CDs (489 nm and 665 nm) through a sequential FRET phenomenon. RhB-CDs were covalently bio-conjugated to probe microRNA. In the presence of AuNPs, the fluorescence of the CDs is quenched, while in the presence of microRNA-155, the ratio of fluorescences at 489 and 665 nm (I489/I665) is enhanced again. A linear relationship exists between the ratio of fluorescence and the concentration of microRNA-155 in the range from 1 aM to 0.1 µM, and the detection limit is 0.3 aM. The assay was applied to quantitative studies of target microRNA-155 in multiple pathways associated with cancer progression in biological fluids include human serum samples and cancer cells. The nanoprobe also deliver clear signal to microRNA target in fixed and lived MDA-MB-231 cells. Graphical abstract A ratiometric FRET sensing method used for microRNA-155 detection at aM concentration level using CDs and AuNPs as donor-acceptor respectively and Rhodamine B as amplification reagent. The application of assay for imaging of microRNA-155 in fixed and live MDA-MB-231 cells is demonstrated.

PyA-cluster system for the detection and imaging of miRNAs in living cells through double-three-way junction formation.

Herein, we describe an extended version of a fluorescence probe for detecting miRNAs through the novel application of a PyA-cluster system. By testing various (CG)n sequences in the middle of the oligonucleotide strand of the probe, we obtained an optimal sequence that formed a double-three-way-junction structure, with two PyA units positioned close together, in the presence of the target miRNA. This system readily detected the locations of target miRNAs in living cells and allowed visualization of structural changes through variations in the color of the fluorescence.