NASBA Coupled to Paper Microfluidics for RNA Detection.

The analysis of RNA sequences is crucial to obtain invaluable insights into disease prognosis. Reliable and rapid diagnostic solutions at the site of sample collection contribute toward optimal delivery of medical treatment. For this reason, the development of more sensitive and portable RNA detection techniques are expected to advance current point-of-care (POC) diagnostic capabilities. Advancements of POC diagnostic technologies will also contribute to counter the spread of emerging viruses. Reverse transcriptase polymerase chain reaction (RT-PCR) is the most commonly used technique to identify etiological organisms of infections. However, the need for thermocycler and fluorescent measurement renders RT-PCR less suitable for POC applications. Here, we provide a step-by-step protocol of Nucleic Acid Sequence-Based Amplification (NASBA), a robust isothermal RNA amplification technique, coupled with a portable paper microfluidics detection format.

Visualization of Individual RNA Molecules by Proximity Ligation-Based Chromogenic In Situ Hybridization Assay.

RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.

RNA Analysis Using Immunoassay Detection Format.

Oligonucleotide probe tagging and reverse transcriptase polymerase-chain reaction (RT-PCR) are the most widely used techniques currently used for detecting and analyzing RNA. RNA detection using labeled oligonucleotide probe-based approaches is suitable for point-of-care (POC) applications but lacks assay sensitivity, whereas RT-PCR requires complex instrumentation. As an alternative, immunoassay detection formats coupled with isothermal RNA amplification techniques have been proposed for handheld assay development. In this chapter, we describe a robust technique comprising of: (a) target RNA tagging with a complementary oligonucleotide probe labeled with a hapten moiety to form a DNA/RNA duplex hybrid; (b) complexing the DNA/RNA duplex with a pre-coated antibody (Ab) directed at the hapten moiety; (c) sandwich complex formation with an Ab that selectively recognizes the DNA/RNA structural motif; and (d) detection of the sandwich complex using a secondary Ab enzyme conjugate targeting the anti-DNA/RNA Ab followed by standard enzyme-linked immunosorbent assay (ELISA) visualization.

Liquid and Solid Hybridization Methods to Detect RNAs.

Northern blotting (NB) has been a long-standing method for RNA detection. However, its labor-intensive nature, reliance on high-quality RNA, and use of radioactivity have diminished its appeal over time. Nevertheless, the emergence of microRNAs (miRNAs) has reignited the demand for sensitive and quantitative NB techniques. We have recently developed cost-effective and rapid protocols for RNA detection using solid and liquid hybridization (LH) techniques which exhibit high sensitivity without the need for radioactive or specialized reagents like locked nucleic acid (LNA) probes. Our assays incorporate biotinylated probes and improved techniques for probe hybridization, transfer, cross-linking, and signal enhancement. We demonstrate that while NB is sensitive in detecting mRNAs and small RNAs, our LH protocol efficiently detects these as well as miRNAs at lower amounts of RNA, achieving higher sensitivity comparable to radiolabeled probes. Compared to NB, LH offers benefits of speed, sensitivity, and specificity in detecting mRNAs, small RNAs, and miRNAs.

Fluorescent Techniques for RNA Detection in Nanoparticles.

The utilization of drug delivery systems, such as lipid nanoparticles and polyplexes/micelleplexes, has shown promise in intracellularly delivering nucleic acids for addressing various diseases. Accurate quantification of the nucleic acid cargo within nanoparticles is essential for the development of safe and effective nanomedicines. Currently, the RiboGreen and SYBR Gold methods are regarded as standard techniques for the precise quantification of RNA in lipid nanoparticles and polyplexes/micelleplexes, respectively. In this chapter, we present a comprehensive protocol for the precise evaluation of the encapsulation efficiency in such formulations using these methods. Additionally, we offer detailed instructions for nanoparticle preparation, characterization, and a comparative analysis of the sensitivity of both methods in quantifying unencapsulated siRNA.

Quantum Dots-Based Protocols for the Detection of RNAs.

RNA (ribonucleic acid) plays a crucial role in various cellular processes and is involved in the development and progression of several diseases. RNA molecules have gained considerable attention as potential biomarkers for various ailments, as they reflect the activity of genes in a particular cell or tissue. By measuring the levels of specific RNA molecules, such as messenger RNA (mRNA), noncoding RNAs, including microRNAs (miRNAs), and long noncoding RNAs (lncRNAs), researchers can infer the expression patterns of genes associated with a particular disease. Aberrant expression of specific miRNAs or lncRNAs has been associated with conditions such as cancer, cardiovascular diseases, neurodegenerative disorders, and more. Detection and quantification of these RNAs in biological samples, such as blood or tissue, can provide valuable diagnostic or prognostic information. Yet their analysis is a challenging endeavor due to their length, sequence similarity across family members, sensitivity to disintegration, and low quantity in total samples. New advances in nanophotonics have provided novel options for fabrication of quantum dots (QDs)-based biosensing devices capable of detecting a variety of disease-specific RNAs. Thus, we proposed and designed a nanophotonic method employing oligonucleotide-conjugated quantum dot nanoconjugates for the rapid and accurate detection of RNAs. Despite the abundance of other molecules in the sample, the approach delivers highly selective, precise identification of the target RNAs. The data also indicated the method's great practicality and simplicity in determining RNAs selectively. Overall, the approach enables the evaluation of RNA expression in relation to the initial onset and progression of a human health disorder.

CRISPR/Cas12a-Powered Amplification-Free RNA Diagnostics by Integrating T7 Exonuclease-Assisted Target Recycling and Split G-Quadruplex Catalytic Signal Output.

Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for RNA detection often rely on reverse transcription (RT) and DNA amplification or involve a time-consuming procedure and poor sensitivity. Herein, we proposed a CRISPR/Cas12a-enabled amplification-free assay for rapid, specific, and sensitive RNA diagnostics. This assay, which we termed T7/G4-CRISPR, involved the use of a T7-powered nucleic acid circuit to convert a single RNA target into numerous DNA activators via toehold-mediated strand displacement reaction and T7 exonuclease-mediated target recycling amplification, followed by activating Cas12a trans-cleavage of the linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion to the input RNA target. We first performed step-by-step validation of the entire assay process and optimized the reaction parameters. Using the optimal conditions, T7/G4-CRISPR was capable of detecting as low as 3.6 pM target RNA, obtaining ∼100-fold improvement in sensitivity compared with the most direct Cas12a assays. Meanwhile, its excellent specificity could discriminate single nucleotide variants adjacent to the toehold region and allow species-specific pathogen identification. Furthermore, we applied it for analyzing bacterial 16S rRNA in 40 clinical urine samples, exhibiting a sensitivity of 90% and a specificity of 100% when validated by RT-quantitative PCR. Therefore, we envision that T7/G4-CRISPR will serve as a promising RNA sensing approach to expand the toolbox of CRISPR-based diagnostics.

Multiplexed in situ RNA imaging by combFISH.

Multiplexed in situ RNA imaging offers new opportunities for gene expression profiling by providing high-throughput spatial information. In this work, we present a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay to achieve multiplexed detection of RNA in cell cultures and tissues. Specifically, multiplexing is achieved through cyclic interrogation of barcode sequences on the rolling circle amplicons generated from the padlock probe assay by using sets of combinatorial detection probes. Theoretically, combFISH can detect 64 genes in three hybridization cycles by combinatorial barcoding using 12 fluorescently labeled detection probes. Our method eliminates sequencing-by-ligation (SBL) chemistry in the in situ sequencing protocol and directly uses RNA as targets for ligation, making it more straightforward. We showed that our method works in fresh-frozen and formalin-fixed paraffin-embedded tissue sections. With its straightforward protocols, we expect our method to be adopted by the scientific community and extended to clinical settings.

Digital Sort-Enabled Counting Allows Absolute Electrical Quantification of Target Nucleic Acid.

Quantitative nucleic acid amplification tests are of great importance for diagnostics, but current approaches require complex and costly optical setups that limit their nonlaboratory applications. Herein we describe the implementation of a microfluidics platform that can perform binary DNA-amplification-activated droplet sorting. The digital sort-enabled counting (DISCO) platform enables label-free absolute quantification of the nucleic acid. This is achieved by provoking a pH change in droplets through a loop-mediated isothermal amplification (LAMP) reaction, followed by using sorting by interfacial tension (SIFT) to direct positive and negative droplets to different outlets. With the use of on-chip electrodes at both outlets, we demonstrate that the digital electrical counting of target DNA and RNA can be realized. DISCO is a promising approach for realizing sensitive nucleic acid quantification in point-of-care settings.

Protocol for high-quality single-cell RNA-seq from tissue sections with DRaqL.

Single-cell RNA sequencing (scRNA-seq) combined with laser capture microdissection (LCM) offers a versatile framework for comprehensive transcriptomics from tissue sections. Here, we present a detailed protocol for DRaqL (direct RNA recovery and quenching for LCM) in combination with Smart-seq2 (DRaqL-Smart-seq2), which enables high-quality RNA sequencing for single cells obtained from alcohol-fixed murine ovarian sections. Additionally, we provide an optional procedure for scRNA-seq from formalin-fixed sections (DRaqL-Protease-Smart-seq2). We outline key steps for cell lysis, cDNA amplification, and sequencing library preparation. For complete details on the use and execution of this protocol, please refer to Ikeda et al.1.

PicoGreen assay for nucleic acid quantification - Applications, challenges, and solutions.

Various analytical methods and reagents have been employed for nucleic acid analysis in cells, biological fluids, and formulations. Standard techniques like gel electrophoresis and qRT-PCR are widely used for qualitative and quantitative nucleic acid analysis. However, these methods can be time-consuming and labor-intensive, with limitations such as inapplicability to small RNA at low concentrations and high costs associated with qRT-PCR reagents and instruments. As an alternative, PicoGreen (PG) has emerged as a valuable method for the quantitative analysis of nucleic acids. PG, a fluorescent dye, enables the quantitation of double-stranded DNA (dsDNA) or double-stranded RNA, including miRNA mimic and siRNA, in solution. It is also applicable to DNA and RNA analysis within cells using techniques like FACS and fluorescence microscopy. Despite its advantages, PG's fluorescence intensity is affected by various experimental conditions, such as pH, salts, and chemical reagents. This review explores the recent applications of PG as a rapid, cost-effective, robust, and accurate assay tool for nucleic acid quantification. We also address the limitations of PG and discuss approaches to overcome these challenges, recognizing the expanding range of its applications.

Effect of Different Staining Methods on Brain Cryosections.

Staining frozen sections is often required to distinguish cell types for spatial transcriptomic studies of the brain. The impact of the staining methods on the RNA integrity of the cells becomes one of the limitations of spatial transcriptome technology with microdissection. However, there is a lack of systematic comparisons of different staining modalities for the pretreatment of frozen sections of brain tissue as well as their effects on transcriptome sequencing results. In this study, four different staining methods were analyzed for their effect on RNA integrity in frozen sections of brain tissue. Subsequently, differences in RNA quality in frozen sections under different staining conditions and their impact on transcriptome sequencing results were assessed by RNA-seq. As one of the most commonly used methods for staining pathological sections, HE staining seriously affects the RNA quality of frozen sections of brain tissue. In contrast, the homemade cresyl violet staining method developed in this study has the advantages of short staining time, low cost, and less RNA degradation. The homemade cresyl violet staining proposed in this study can be applied instead of HE staining as an advance staining step for transcriptome studies in frozen sections of brain tissue. In the future, this staining method may be suitable for wide application in brain-related studies of frozen tissue sections. Moreover, it is expected to become a routine step for staining cells before sampling in brain science.

Simultaneous Detection of Adenosine-to-Inosine Editing and N6-Methyladenosine at Identical RNA Sites through Deamination-Assisted Reverse Transcription Stalling.

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) modifications are pivotal RNA modifications with widespread functional significance in physiological and pathological processes. Although significant effort has been dedicated to developing methodologies for identifying and quantifying these modifications, traditional approaches have often focused on each modification independently, neglecting the potential co-occurrence of A-to-I editing and m6A modifications at the same adenosine residues. This limitation has constrained our understanding of the intricate regulatory mechanisms governing RNA function and the interplay between different types of RNA modifications. To address this gap, we introduced an innovative technique called deamination-assisted reverse transcription stalling (DARTS), specifically designed for the simultaneous quantification of A-to-I editing and m6A at the same RNA sites. DARTS leverages the selective deamination activity of the engineered TadA-TadA8e protein, which converts adenosine residues to inosine, in combination with the unique property of Bst 2.0 DNA polymerase, which stalls when encountering inosine during reverse transcription. This approach enables the accurate quantification of A-to-I editing, m6A, and unmodified adenosine at identical RNA sites. The DARTS method is remarkable for its ability to directly quantify two distinct types of RNA modifications simultaneously, a capability that has remained largely unexplored in the field of RNA biology. By facilitating a comprehensive analysis of the co-occurrence and interaction between A-to-I editing and m6A modifications, DARTS opens new avenues for exploring the complex regulatory networks modulated by different RNA modifications.

Contact lenses as novel tear fluid sampling vehicles for total RNA isolation, precipitation, and amplification.

The tear fluid is a readily accessible, potential source for biomarkers of disease and could be used to monitor the ocular response to contact lens (CL) wear or ophthalmic pathologies treated by therapeutic CLs. However, the tear fluid remains largely unexplored as a biomarker source for RNA-based molecular analyses. Using a rabbit model, this study sought to determine whether RNA could be collected from commercial CLs and whether the duration of CL wear would impact RNA recovery. The results were referenced to standardized strips of filtered paper (e.g., Shirmer Strips) placed in the inferior fornix. By performing total RNA isolation, precipitation, and amplification with commercial kits and RT-PCR methods, CLs were found to have no significant differences in RNA concentration and purity compared to Schirmer Strips. The study also identified genes that could be used to normalize RNA levels between tear samples. Of the potential control genes or housekeeping genes, GAPDH was the most stable. This study, which to our knowledge has never been done before, provides a methodology for the detection of RNA and gene expression changes from tear fluid that could be used to monitor or study eye diseases.

Chemical Annealing Restructures RNA for Nanopore Detection.

RNA is a key biochemical marker, yet its chemical instability and complex secondary structure hamper its integration into DNA nanotechnology-based sensing platforms. Relying on the denaturation of the native RNA structure using urea, we show that restructured DNA/RNA hybrids can readily be prepared at room temperature. Using solid-state nanopore sensing, we demonstrate that the structures of our DNA/RNA hybrids conform to the design at the single-molecule level. Employing this chemical annealing procedure, we mitigate RNA self-cleavage, enabling the direct detection of restructured RNA molecules for biosensing applications.

Evaluation of short-term hair follicle storage conditions for maintenance of RNA integrity.

Hair follicles provide an easily accessible tissue for interrogating gene expression for multiple purposes in mammals. RNAlater® is a liquid storage solution that stabilises and preserves cellular RNA, eliminating the need to immediately process or freeze tissue specimens. The manufacturer advises storage of samples at 2-8°C overnight before transfer to -20°C. This study aimed to evaluate RNA integrity in hair follicle samples collected from horses, stabilized in RNAlater®, and stored under three short-term storage conditions. Mane hair samples complete with follicles were collected from four horses at a single time point. Approximately 15 hairs were placed in each of three 2 mL tubes containing 0.75ml RNAlater® solution. Test group A was stored at 4°C for 24-h, then decanted and stored at -20°C. Test groups B and C were stored at 4°C and 19°C (room temperature) respectively for 7 days, then decanted and stored at -20°C. RNA was isolated from all samples and RNA quantity and quality were measured. One-way ANOVA revealed no difference in RNA concentration (A:516 +/-125 ng/ml, B:273+/-93 ng/ml, C:476+/-176 ng/ml;P = 0.2) or quality (A:9.5 +/-0.19, B:9.8+/-0.09, C:9.2+/-0.35 RIN; P = 0.46) between the test groups. There were no group differences in mean Cycle Threshold values from qPCR validation assays confirming high-quality template cDNA. The results suggest that storage of hair follicles for one week in RNAlater® at cool or room temperature conditions will not compromise RNA integrity and will permit extended transport times from remote sampling locations without the need for freezing.

Construction of a 3D Quantum Dot Nanoassembly with Two-Step FRET for One-Step Sensing of Human Telomerase RNA in Breast Cancer Cells and Tissues.

Telomerase is an important biomarker for early diagnosis of cancers, but current telomerase assays usually rely on measuring the extension products of telomerase substrates, which increases the assay complexity. More evidence indicates that human telomerase RNA (hTR), as a core component of telomerase, is positively correlated with the telomerase activity. Herein, we demonstrate the development of a duplex-specific nuclease (DSN)-propelled 3D quantum dot (QD) nanoassembly with two-step Föster resonance energy transfer (FRET) for the one-step sensing of hTR in breast cancer cells and tissues. This assay involves only one hairpin probe modified with a Cy5 at the sixth base from the 5'-biotin end and a BHQ2 at the 3'-terminus, which integrates three functions of target recognition, target recycling amplification, and signal readout. The anchoring of the hairpin probe on the 605QD surface results in the formation of a 3D 605QD-Cy5-probe-BHQ2 nanoassembly in which two-step FRET occurs among the 605QD, Cy5, and BHQ2 quencher. Notably, the formation of 605QD-Cy5-probe-BHQ2 nanoassembly facilitates the reduction of background signal and the increase of signal-to-background ratio due to its dense, highly oriented nucleic acid shell-induced steric hindrance effect. This assay can achieve one-step and rapid detection of hTR with a detection limit of 2.10 fM, which is the simplest and most rapid hTR assay reported so far. Moreover, this assay can efficiently distinguish single-base mismatched sequences, and it can discriminate the hTR level between breast cancer patients and healthy donors with a high accuracy of 100%, with great prospects for early diagnosis of cancers.

Discrimination of Genetic Biomarkers of Disease through Machine-Learning-Based Hypothesis Testing of Direct SERS Spectra of DNA and RNA.

Cancer is globally a leading cause of death that would benefit from diagnostic approaches detecting it in its early stages. However, despite much research and investment, cancer early diagnosis is still underdeveloped. Owing to its high sensitivity, surface-enhanced Raman spectroscopy (SERS)-based detection of biomarkers has attracted growing interest in this area. Oligonucleotides are an important type of genetic biomarkers as their alterations can be linked to the disease prior to symptom onset. We propose a machine-learning (ML)-enabled framework to analyze complex direct SERS spectra of short, single-stranded DNA and RNA targets to identify relevant mutations occurring in genetic biomarkers, which are key disease indicators. First, by employing ad hoc-synthesized colloidal silver nanoparticles as SERS substrates, we analyze single-base mutations in ssDNA and RNA sequences using a direct SERS-sensing approach. Then, an ML-based hypothesis test is proposed to identify these changes and differentiate the mutated sequences from the corresponding native ones. Rooted in "functional data analysis," this ML approach fully leverages the rich information and dependencies within SERS spectral data for improved modeling and detection capability. Tested on a large set of DNA and RNA SERS data, including from miR-21 (a known cancer miRNA biomarker), our approach is shown to accurately differentiate SERS spectra obtained from different oligonucleotides, outperforming various data-driven methods across several performance metrics, including accuracy, sensitivity, specificity, and F1-scores. Hence, this work represents a step forward in the development of the combined use of SERS and ML as effective methods for disease diagnosis with real applicability in the clinic.

Reliable detection of RNA in hippocampus sections of mice by FISH up to a post-mortem delay of 24 h.

Proteins can be successfully localized in post-mortem (PM) brain tissue sections if the time until PM tissue sampling is not too long. In this study, we show that this also applies to the localization of RNA and in particular to the RNA of microglia-specific receptor proteins using the probes and the RNAscope™ Multiplex Fluorescent Detection Kit v2 from Advanced Cell Diagnostics. Brains were removed from killed mice after different PM delays and processed into paraffin sections. In sections of brains from animals whose cadavers had been kept at room temperature (21 °C) before tissue removal, ubiquitously expressed RNAs of genes with low to high expression levels (Polr2a, PPIB, and UBC) were reliably detected in the brain sections even if tissue removal was delayed by up to 48 h. In addition, microglia-specific G protein-coupled receptor RNA (Gpr34, P2ry12) could be reliably assigned to microglia by simultaneous labeling of the microglia with microglia-specific antibodies (Iba1 or P2ry12). Only after a delay of 48 h until tissue removal were the receptor RNA signals significantly lower. The reduction in receptor RNA signals could be delayed if the animal cadavers were stored at 4 °C until the brains were removed. Tissue sections of PM brain samples allow the spatial and cellular localization of specific RNA, at least if the sampling takes place within the first 24 h of PM.

In-vitro Clinical Diagnostics using RNA-Cleaving DNAzymes.

Over the last three decades, significant advancements have been made in the development of biosensors and bioassays that use RNA-cleaving DNAzymes (RCDs) as molecular recognition elements. While early examples of RCDs were primarily responsive to metal ions, the past decade has seen numerous RCDs reported for more clinically relevant targets such as bacteria, cancer cells, small metabolites, and protein biomarkers. Over the past 5 years several RCD-based biosensors have also been evaluated using either spiked biological matrixes or patient samples, including blood, serum, saliva, nasal mucus, sputum, urine, and faeces, which is a critical step toward regulatory approval and commercialization of such sensors. In this review, an overview of the methods used to generate RCDs and the properties of key RCDs that have been utilized for in vitro testing is first provided. Examples of RCD-based assays and sensors that have been used to test either spiked biological samples or patient samples are then presented, highlighting assay performance in different biological matrixes. A summary of current prospects and challenges for development of in vitro diagnostic tests incorporating RCDs and an overview of future directions of the field is also provided.