Search

MicroRNAs play a crucial role in regulating plant growth, such as involving in the pathways of phytohormons signaling, which function in broad range of important aspects in many stages of plant development and evolution. To figure out the components part of microRNA in phytohormone signaling network, a sensitive photoelectrochemical biosensor was developed to detect miRNA-319a specifically using CuO-CuWO 4 as photoactive material and in situ generation electron donor technique. Taking the advantage of rolling circle amplification, nicking endonuclease triggered exponential amplification of target molecule, specific interaction between phosphate group and phos-tag and between biotin and avidin, the developed strategy can greatly amplify the detection signal only using trace amount of target miRNA-319a. The wide linear range was obtained from 1 fM to 0.1 nM and the detection limit was estimated to be 0.47 fM (S/N = 3). The developed method also showed good detection specificity. The effect of five phytohormones on the expression level of miRNA-319a in rice leaves was also investigated. This will be a basal principle of investigating the signaling network of phytohormones. [ABSTRACT FROM AUTHOR]

It is extremely important for quantifying trace microRNAs in the biomedical applications. In this study, an ultrasensitive, rapid and efficient label-free fluorescence method was proposed and applied for detecting microRNA-21 in serum of gastric cancer patients based on DNA hybridization chain reaction (HCR). DNA H1 and DNA H2 were designed and used as hairpin probes, the HCR was proceeded in the presence of target microRNAs. Amounts of SYBR Green І dyes were used as signal molecules to intercalate long DNA concatemers from HCR, which guaranteed the model of label-free fluorescence and strong fluorescence density. The detection method showed a wide linear region from 1 fM to 10 5 fM, and the limit of detection was 0.2554 fM (at S/N = 3) for microRNAs. The results showed that this method had an excellent specificity and reproducibility. Furthermore, the label-free fluorescence strategy exhibited a sensitive response to microRNA-21 in real serum samples of gastric cancer patients and the results obtained were in accordance with reference method (R 2 = 0.994). Overall, the proposed strategy could be satisfactory for rapid, ultrasensitive and efficient detection of microRNA-21, and held great potentials in clinic diagnosis of gastric cancer. [ABSTRACT FROM AUTHOR]

The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) is riveting nowadays due to its close relevance to human malignancies. Exploiting a fast and convenient biosensor for sensitive and specific detecting miRNA is necessary and it has significant meaning for oncology. In this work, to understand the relationship between miRNA-21 and gastric cancer, we employ T4 RNA ligase 2 to initiate specific ligation reaction depending on the sequence of target RNA, and T7 exonuclease to catalyze the first stage cyclic enzymatic amplification method (CEAM) specifically, which also resting on the target RNA sequence, and the second stage CEAM to further amplify the response resulting from the initial target RNA. Our two-stage CEAM strategy can detect miRNA-21 as low as 0.36 fM with remarkable specificity, and most importantly, it can be used to inspect the expression level of miRNA-21 in blood serum of gastric cancer patients. This will offer new perspective for figuring out the pathways of miRNA-21 involving in cancer. [ABSTRACT FROM AUTHOR]

In this work, we proposed a selective and sensitive biosensor for microRNA detection based on the high specificity and affinity of anti-DNA:RNA hybrids antibody (S9.6 antibody) and alkaline phosphatase catalytic signal amplification. Briefly, after the hybridization of probe DNA and the target microRNA, the S9.6 antibody can be captured on the electrode surface through antigen-antibody immunoreaction. Then, alkaline phosphatase labeled goat anti-mouse IgG (ALP-IgG) was further captured on the electrode surface through the specific recognition effect between the primary antibody and the secondary antibody. Finally, ALP catalyzed the hydrolysis reaction of p -nitrophenyl phosphate to generate p -nitrophenol, resulting in a electrochemical oxidation signal. The simple signal amplification assay performed a successful linear range from 0.5-500 fM with a detection limit of 0.40 fM. Moreover, this biosensor exhibited high selectivity with discriminating only single-base mismatched microRNA sequence. Additionally, the simplicity of this method hold a great promise for further investigation of the role of microRNAs in phytohormone signaling transduction. [ABSTRACT FROM AUTHOR]

A selective and sensitive photoelectrochemical assay was proposed for microRNA detection based on the isothermal cycle hybridization amplification of microRNA-21 that originated from the specific cleavage activity of duplex-specific nuclease (DSN) toward a DNA probe in DNA-RNA double helix and in situ enzymatic production of electron donor of ascorbic acid. After the biotin-functionalized DNA probe hybridized with complementary target microRNA-21, the hybridized DNA probe could be cleaved by DSN and microRNA-21 was released back to the incubation solution. The released microRNA could be further hybridized with the remaining single-strand DNA probe; thus, the isothermal cycle hybridization amplification would be formed. As a result, little avidin-alkaline phosphatase conjugate could be captured due to the release of biotin originating from the DSN cleavage. The distinct photocurrent change between a control biosensor and the DSN cleavage biosensor achieved label-free microRNA-21 detection with the linear range from 1 to 500 fM. The fabricated biosensor showed high detection selectivity even for one-base mismatched sequence. The attempt was carried out to directly assay the expression level change of microRNA-21 in total RNA extracted from chicken fibroblast cells infected with subgroup J avian leukosis virus. [ABSTRACT FROM AUTHOR]

Herein, a sensitive and selective photoelectrochemical (PEC) immunoassay is developed for microRNA, where the graphite-like C 3 N 4 (g-C 3 N 4 ) is used as photoactive material and anti-DNA:RNA antibody is used as target microRNA recognition unit. With the doping of gold nanoparticles (AuNPs), the photoactivity of g-C 3 N 4 is greatly improved. The capture probe for microRNA is assembled on the AuNPs-g-C 3 N 4 modified ITO surface through the Au S bond. After hybridizing with target microRNA, the DNA:RNA hybrids can be recognized by anti-DNA:RNA antibody and this antibody can be further captured on the electrode surface. Through the specific interaction between antibody and alkaline phosphatase labeled IgG (ALP-IgG), ALP-IgG can be captured, which can catalyze the hydrolysis of ascorbic acid-2-phosphate (AAP) to produce ascorbic acid as electron donor. As a result, a strong photocurrent is obtained and the expression level of microRNA can be detected based the change of photocurrent. The fabricated biosensor presents good detection sensitivity with the linear range of 5–3000 fM and the detection limit of 2.26 fM. The developed biosensor also shows accepted detection selectivity with identifying even single-base mismatched microRNA. The applicability of the biosensor is further evaluated by assaying the expression level change of microRNA in rice leaves with and without phytohormone incubation. This work is not only providing a new detection platform for microRNA assay, but also affording some information on the role of microRNA in phytohormone signaling. [ABSTRACT FROM AUTHOR]

MicroRNAs play crucial role in post-transcriptional regulation for gene expression in animals, plants, and viruses. For the better understanding of microRNA and its functions, it is very important to develop effectively analytical method for microRNA detection. Herein, a novel electrochemical biosensor was fabricated for sensitive and selective detection of microRNA based on poly(U) polymerase mediated isothermal signal amplification, where poly(U) polymerase can catalyze the template independent addition of UMP from UTP to the 3’ end of RNA. Using this activity, the target microRNA can be successfully labeled with biotin conjugated UMPs at its 3′-end using biotin conjugated UTP (biotin-UTP) as donor. Then, the avidin conjugated alkaline phosphatase can be further captured to the 3′-end of the target microRNA based on the specific interaction between biotin and avidin. Finally, under the catalytic activity of alkaline phosphatase, the substrate of p -nitrophenyl phosphate disodium salt hexahydrate can be hydrolyzed to produce 4-nitrophenol. According to the relationship between the electrochemical signal of p -nitrophenol and the concentration of microRNA-319a, the content of microRNA-319a can be detected. This signal amplification method is simple and sensitive. The developed method can detect as low as 1.7 fM microRNA and produce precise and accurate linear dynamic range from 10 to 1000 fM. The fabricated biosensor was further applied to detect the expression level change of microRNA-319a in rice seedlings after incubation with five kinds of different phytohormones. [ABSTRACT FROM AUTHOR]