Detection Of Small RNAs Research Articles

Detection of small noncoding RNAs by in situ hybridization using probes of 2'-O-methyl RNA + LNA.

In situ hybridization is a powerful method to provide information about contextual distribution and cellular origin of nucleic acids, e.g., in formalin-fixed paraffin-embedded (FFPE) samples of tissue. Particularly the recently discovered classes of noncoding RNA (ncRNA) including endo-siRNAs and microRNAs require such a technique to enable their study and visualization in natural contexts, and in the last decade, many advances have been made, increasing our ability to specifically detect small ncRNAs. One of the key developments has been the demonstration of the superiority of using locked nucleic acid (LNA)-modified DNA probes for the detection of ncRNA in tissue. Here, we describe an alternative in situ hybridization protocol employing oligonucleotide probes consisting of combinations of LNA and 2´-O-methyl RNAs that under optimized hybridization buffer conditions can provide a highly sensitive assay performance with only 1 h hybridization time.

Counting Small RNA in Pathogenic Bacteria

Here, we present a modification to single-molecule fluorescence in situ hybridization that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short (∼200 nucleotide) nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. By neutralizing the fluorescence from unbound probes, we were able to significantly reduce the number of false positives, allowing for accurate quantification of sRNA levels. Exploiting an automated, mutli-color wide-field microscope and data analysis package, we analyzed the statistics of sRNA expression in thousands of individual bacteria. We found that only a small fraction of either Yersinia pseudotuberculosis or Yersinia pestis bacteria express the small RNAs YSR35 or YSP8, with the copy number typically between 0 and 10 transcripts. The numbers of these RNA are both increased (b...

Extraction and nonradioactive detection of small RNA molecules.

The emergence of small RNAs as key and potent regulators of gene expression has prompted the need for robust detection and assay protocols to be developed for investigating their generation and tissue distribution. The physicochemical nature of these RNAs allows traditional assay methods to be employed; however, due to the relatively small size of endo-siRNAs, key changes to these protocols are required. Here, we present a method for the nonradioactive detection of endo-siRNAs in mouse tissue and microinjected Xenopus oocytes. The method comprises steps for RNA extraction, PAGE, and low-stringency northern blotting using DIG-labelled RNA probes. Moreover, it includes a strategy to design and generate cheap hybridization probes with greatly increased sensitivity. These methods may be used as a simple and robust protocol for nonradioactive detection of small RNAs or be combined with other strategies to potentially enhance signal intensity.

Enhanced detection of small RNAs using a nonradioactive approach.

Recent advancements in high-throughput sequencing have led to the identification of many new classes of small noncoding RNAs such as endo-siRNAs. Unfortunately, reliable quantification of RNAs by sequencing is difficult due to artifacts arising from various factors involved in cDNA library preparation. Northern blot is one of the leading methods used to confirm the presence of a given RNA sequence because it can accurately quantify the cellular abundance, the size of the small RNA and reveal the presence of potential precursors and RNA isoforms. Here, we present a comprehensive description of LNA probe design along with a recently developed highly sensitive and cost-effective nonradioactive northern blot approach termed LED. LED combines a cross-linking method (EDC) and digoxigenin (DIG) labeling, and it can detect small RNAs with concentrations as low as 0.05 fmol and requires as little as a few seconds of membrane exposure for signal generation.

A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA

Several methods for the detection of RNA have been developed over time. For small RNA detection, a stem-loop reverse primer-based protocol relying on TaqMan RT-PCR has been described. This protocol requires an individual specific TaqMan probe for each target RNA and, hence, is highly cost-intensive for experiments with small sample sizes or large numbers of different samples. We describe a universal TaqMan-based probe protocol which can be used to detect any target sequence and demonstrate its applicability for the detection of endogenous as well as artificial eukaryotic and bacterial small RNAs. While the specific and the universal probe-based protocol showed the same sensitivity, the absolute sensitivity of detection was found to be more than 100-fold lower for both than previously reported. In subsequent experiments, we found previously unknown limitations intrinsic to the method affecting its feasibility in determination of mature template RISC incorporation as well as in multiplexing. Both protocols were equally specific in discriminating between correct and incorrect small RNA targets or between mature miRNA and its unprocessed RNA precursor, indicating the stem-loop RT-primer, but not the TaqMan probe, triggers target specificity. The presented universal TaqMan-based RT-PCR protocol represents a cost-efficient method for the detection of small RNAs.

Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicumATCC 13032

BackgroundRecent discoveries on bacterial transcriptomes gave evidence that small RNAs (sRNAs) have important regulatory roles in prokaryotic cells. Modern high-throughput sequencing approaches (RNA-Seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. Whole transcriptome data obtained by RNA-Seq can be used to detect and characterize all transcript species, including small RNAs. Here, we describe an RNA-Seq approach for comprehensive detection and characterization of small RNAs from Corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important Corynebacterianeae, such as C. diphtheriae and Mycobacterium tuberculosis.ResultsIn our RNA-Seq approach, total RNA from C. glutamicum ATCC 13032 was prepared from cultures grown in minimal medium at exponential growth or challenged by physical (heat shock, cold shock) or by chemical stresses (diamide, H2O2, NaCl) at this time point. Total RNA samples were pooled and sequencing libraries were prepared from the isolated small RNA fraction. High throughput short read sequencing and mapping yielded over 800 sRNA genes. By determining their 5′- and 3′-ends and inspection of their locations, these potential sRNA genes were classified into UTRs of mRNAs (316), cis-antisense sRNAs (543), and trans-encoded sRNAs (262). For 77 of trans-encoded sRNAs significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species C. efficiens YS-314 and C. diphtheriae NCTC 13129. Three selected trans-encoded sRNAs were characterized by Northern blot analysis and stress-specific transcript patterns were found.ConclusionsThe study showed comparable numbers of sRNAs known from genome-wide surveys in other bacteria. In detail, our results give deep insight into the comprehensive equipment of sRNAs in C. glutamicum and provide a sound basis for further studies concerning the functions of these sRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-714) contains supplementary material, which is available to authorized users.

Complete genomic sequence of a Rubus yellow net virus isolate and detection of genome-wide pararetrovirus-derived small RNAs

Rubus yellow net virus (RYNV) was cloned and sequenced from a red raspberry (Rubus idaeus L.) plant exhibiting symptoms of mosaic and mottling in the leaves. Its genomic sequence indicates that it is a distinct member of the genus Badnavirus, with 7932bp and seven ORFs, the first three corresponding in size and location to the ORFs found in the type member Commelina yellow mottle virus. Bioinformatic analysis of the genomic sequence detected several features including nucleic acid binding motifs, multiple zinc finger-like sequences and domains associated with cellular signaling. Subsequent sequencing of the small RNAs (sRNAs) from RYNV-infected R. idaeus leaf tissue was used to determine any RYNV sequences targeted by RNA silencing and identified abundant virus-derived small RNAs (vsRNAs). The majority of the vsRNAs were 22-nt in length. We observed a highly uneven genome-wide distribution of vsRNAs with strong clustering to small defined regions distributed over both strands of the RYNV genome. Together, our data show that sequences of the aphid-transmitted pararetrovirus RYNV are targeted in red raspberry by the interfering RNA pathway, a predominant antiviral defense mechanism in plants.

FCGR2A and FCGR3A polymorphisms in classical Hodgkin lymphoma by Epstein–Barr virus status

FCGR2A and FCGR3A polymorphisms in classical Hodgkin lymphoma by Epstein–Barr virus status

Small nucleolar RNA-derived microRNA hsa-miR-1291 modulates cellular drug disposition through direct targeting of ABC transporter ABCC1.

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is an important membrane transporter that contributes to cellular disposition of many endobiotic and xenobiotic agents, and it can also confer multidrug resistance. This study aimed to investigate the role of human noncoding microRNA-1291 (hsa-miR-1291) in regulation of ABCC1 and drug disposition. Bioinformatics analyses indicated that hsa-miR-1291, localized within the small nucleolar RNA H/ACA box 34 (SNORA34), might target ABCC1 3'-untranslated region (3'UTR). Using splinted ligation small RNA detection method, we found that SNORA34 was processed into hsa-miR-1291 in human pancreatic carcinoma PANC-1 cells. Luciferase reporter assays showed that ABCC1 3'-UTR-luciferase activity was decreased by 20% in cells transfected with hsa-miR-1291 expression plasmid, and increased by 40% in cells transfected with hsa-miR-1291 antagomir. Furthermore, immunoblot study revealed that ABCC1 protein expression was sharply reduced in hsa-miR-1291-stably transfected PANC-1 cells, which was attenuated by hsa-miR-1291 antagomir. The change of ABCC1 protein expression was associated with an alternation in mRNA expression. In addition, hsa-miR-1291-directed downregulation of ABCC1 led to a greater intracellular drug accumulation and sensitized the cells to doxorubicin. Together, our results indicate that hsa-miR-1291 is derived from SNORA34 and modulates cellular drug disposition and chemosensitivity through regulation of ABCC1 expression. These findings shall improve the understanding of microRNA-controlled epigenetic regulatory mechanisms underlying multidrug resistance and interindividual variability in pharmacokinetics.

Counting Small RNA in Pathogenic Bacteria

Here, we present a modification to single-molecule fluorescence in situ hybridization that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short (~200 nucleotide) nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. By neutralizing the fluorescence from unbound probes, we were able to significantly reduce the number of false positives, allowing for accurate quantification of sRNA levels. Exploiting an automated, mutli-color wide-field microscope and data analysis package, we analyzed the statistics of sRNA expression in thousands of individual bacteria. We found that only a small fraction of either Yersinia pseudotuberculosis or Yersinia pestis bacteria express the small RNAs YSR35 or YSP8, with the copy number typically between 0 and 10 transcripts. The numbers of these RNA are both increased (by a factor of 2.5× for YSR35 and 3.5× for YSP8) upon a temperature shift from 25 to 37 °C, suggesting they play a role in pathogenesis. The copy number distribution of sRNAs from bacteria-to-bacteria are well-fit with a bursting model of gene transcription. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in cellular regulatory networks.

Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis

Development of a rapid and accurate quantification method for the detection of microRNAs (miRNAs) has been desired, in particular, when they are differently expressed in normal and pathological conditions. However, various methods for the quantification of small non-coding RNAs as well as miRNAs have been described. These methods mainly include hybridization-based approaches such as primer extension, northern blotting, microarray profiling, and reverse transcription (RT) PCR. Here, we developed a simple and rapid method based on stem-loop primer-based real-time PCR assay for sensitive and accurate detection of mature miRNAs. Initially, a miRNA-specific stem-loop RT primer is used for RT, which is followed by TaqMan real-time PCR assay using specific forward primer in combination with universal reverse primer and TaqMan probe. The assay has shown high sensitivity (≤50 copies/reaction) for miRNA detection in two breast cancer cell lines, MCF-7 and MDA-MB-231. This assay might be implicated as a rapid and cost effective method for the detection of small non-coding RNAs.

A High Resolution Short Interfering RNA (siRNA) Detection Method from Virus-infected Plants

[Abstract] Plant viruses are strong inducers as well as targets of RNA silencing. In plants RNA silencing acts as a natural defense mechanism against viral infection and is associated with accumulation of virus-specific small interfering RNAs (siRNAs). The continuing discoveries, increasing awareness and interest in the regulatory roles of non-coding small RNAs have raised the need for methods that can reliably detect and quantitate the expression levels of small RNAs. Northern blot analysis of small RNAs involving the separation of RNA molecules using polyacrylamide gel electrophoresis (PAGE) has remained a popular and valuable analytical method to validate small RNAs. Northern blot analysis consist of resolving RNAs by gel electrophoresis, followed by transferring and fixing to nylon membranes as well as detecting by hybridization using radioactive probes. The following protocol provides a method for isolation and detection of small RNAs from virus-infected plants and was successfully used in Panwar et al. (2013a), Panwar et al. (2013b).

Splinted Ligation Method to Detect Small RNAs

In any cell, the number of RNA species is remarkably complex. The sizes of RNAs can vary from 20 nucleotides to several kilobases, and abundances can vary from a few to hundreds of thousands of molecules per cell. It is of obvious interest to determine the abundance and integrity of specific RNA species within these complex mixtures. This protocol describes the splinted ligation method to detect small RNAs. It relies on the ability of T4 DNA ligase to covalently join the terminal 3'-hydroxyl group of an RNA molecule to the labeled 5'-phosphate group of a DNA chain in the presence of a DNA "splint" or "bridge" oligonucleotide that is complementary to both. After ligation, the labeled small RNA, lengthened by the covalent addition of the (32)P-labeled oligonucleotide probe, is visualized by denaturing gel electrophoresis and phosphorimaging. This approach is recommended for the routine detection and quantification of specific small RNAs (e.g., microRNAs [miRNAs] and piwi-associated RNAs [piRNAs]).

P141 Viral genome sequencing and small RNA detection by next generation sequencing

Introduction Next generation sequencing technologies are increasingly used in many domains. Their application to virology opens the door to various investigations such as virus sequencing and genotyping; analysis at quasispecies level; identification of causative agents of undiagnosed diseases; and finally study of host responses induced by viral infections. In this study, we used influenza- and rhinoviruses as models to validate some of these applications. Methods First we used influenza A virus infected cells to compare complete genome coverage after purification and ultra-deep sequencing of small RNAs (20–30 nt) or total fragmented RNAs (150–250 nt). Second, we sequenced small RNA population in rhinovirus (HRB-B14 and HRV-A39) infected HeLa cells and analyzed human micro RNA (miRNA) changes and the distribution of sequence reads on the viral genome. Results As expected the number of viral reads and genome coverage were much higher by sequencing total RNA, despite a lower ratio of viral versus host sequences. In contrast, small RNA purification from rhinovirus infected cells allowed us to point out to miRNA expression changes upon viral infection as well as over-expressed small viral RNAs (svRNAs). Conclusion svRNAs match the rhinovirus 5′UTR cloverleaf region, an important structural element implicated in the regulation of replication. The generation and the functional role of these svRNAs is currently under investigation.

Locked Nucleic Acid Flow Cytometry-fluorescence <em>in situ</em> Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence <em>in situ</em> Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid and Flow Cytometry-Fluorescence In Situ Hybridization for the Detection of Bacterial Small Noncoding RNAs

We describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescence in situ hybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) in Vibrio campbellii ATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables the in situ detection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population.

Searching for biomarkers in a historic sample of Amyotrophic Lateral Sclerosis – The Munich Historic Brain Bank Project

Post-mortem analysis of CNS tissue was the gold-standard in the diagnosis of neurologic and psychiatric diseases in former times. This resulted in the storage of hundreds of thousands of precisely catalogued brain tissue specimens at our institute. As a first approach towards using these specimens for modern reanalysis, we identified 62 patients with clinical definite or probable Amyotrophic Lateral Sclerosis (ALS). After diagnostic verification, we successfully established modified methods for the isolation of genomic DNA from formalin-fixed, paraffin-embedded (FFPE) CNS tissue from glass object carriers. Despite the strong grade of DNA degradation and fragmentation, we were able to detect a variety of single nucleotide polymorphisms (SNP) which were shown to be significantly associated with ALS in recent genome-wide association studies (GWAS). 11 SNPs seem to show potential significant association in our patient sample compared to 123 neurologically healthy controls with 3 SNPs bearing p-values < 0.01. However, call rates differed to a considerable degree given the low quality of the preserved nucleic acids. Additionally, we work on standardized methods for the isolation and detection of small non-coding RNAs to take advantage of the easily accessable brain tissue. Our MPI-P historic brain bank offers unique opportunities not only for the verification but also for the discovery of new biomarkers in psychiatric or neurologic diseases.

MirExplorer: Detecting microRNAs from genome and next generation sequencing data using the AdaBoost method with transition probability matrix and combined features

microRNAs (miRNAs) represent an abundant group of small regulatory non-coding RNAs in eukaryotes. The emergence of Next-generation sequencing (NGS) technologies has allowed the systematic detection of small RNAs (sRNAs) and de novo sequencing of genomes quickly and with low cost. As a result, there is an increased need to develop fast miRNA prediction tools to annotate miRNAs from various organisms with a high level of accuracy, using the genome sequence or the NGS data. Several miRNA predictors have been proposed to achieve this purpose. However, the accuracy and fitness for multiple species of existing predictors needed to be improved. Here, we present a novel prediction tool called mirExplorer, which is based on an integrated adaptive boosting method and contains two modules. The first module named mirExplorer-genome was designed to de novo predict pre-miRNAs from genome, and the second module named mirExplorer-NGS was used to discover miRNAs from NGS data. A set of novel features of pre-miRNA secondary structure and miRNA biogenesis has been extracted to distinguish real pre-miRNAs from pseudo ones. We used outer-ten-fold cross-validation to verify the mirExplorer-genome computation, which obtained a specificity of 95.03% and a sensitivity of 93.71% on human data. This computation was made on test data from 16 species, and it achieved an overall accuracy of 95.53%. Systematic outer-ten-fold cross-validation of the mirExplorer-NGS model achieved a specificity of 98.3% and a sensitivity of 97.72%. We found that the good performance of the mirExplorer-NGS model was upheld across species from vertebrates to plants in test datasets. The mirExplorer is available as both web server and software package at http://biocenter.sysu.edu.cn/mir/.

Micro-RNA quantification using DNA polymerase and pyrophosphate quantification

A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20–100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays.