Complementary miRNA Research Articles

Knockdown of Simulated-Solar-Radiation-Sensitive miR-205-5p Does Not Induce Progression of Cutaneous Squamous Cell Carcinoma In Vitro.

Solar radiation is the main risk factor for cSCC development, yet it is unclear whether the progression of cSCC is promoted by solar radiation in the same way as initial tumorigenesis. Additionally, the role of miRNAs, which exert crucial functions in various tumors, needs to be further elucidated in the context of cSCC progression and connection to solar radiation. Thus, we chronically irradiated five cSCC cell lines (Met-1, Met-4, SCC-12, SCC-13, SCL-II) with a custom-built irradiation device mimicking the solar spectrum (UVB, UVA, visible light (VIS), and near-infrared (IRA)). Subsequently, miRNA expression of 51 cancer-associated miRNAs was scrutinized using a flow cytometric multiplex quantification assay (FirePlex®, Abcam). In total, nine miRNAs were differentially expressed in cell-type-specific as well as universal manners. miR-205-5p was the only miRNA downregulated after SSR-irradiation in agreement with previously gathered data in tissue samples. However, inhibition of miR-205-5p with an antagomir did not affect cell cycle, cell growth, apoptosis, or migration in vitro despite transient upregulation of oncogenic target genes after miR-205-5p knockdown. These results render miR-205-5p an unlikely intracellular effector in cSCC progression. Thus, effects on intercellular communication in cSCC or the simultaneous examination of complementary miRNA sets should be investigated.

Molecular modeling of PARP1 inhibition through miRNA gene silencing for the treatment of retinitis pigmentosa.

Retinitis Pigmentosa (RP) is a category of diseases that are characterized by the death of rod photoreceptors and subsequent death of cone receptors. Parthanatos, a form of regulated necrosis, has been reported as being the underlying mechanism of cell death in RP. Parthanatos in RP occurs with the downstream expression of poly-ADP-ribose-polymerase (PARP) and the subsequent nuclear translocation of the mitochondrial apoptosis-inducing factor (AIF), leading to condensing of chromatin and DNA fragmentation. Extracellular vesicles (EVs) are small-scale liposomes that carry miRNAs and the RNA-induced silencing complex (RISC) associated with gene regulation. miRNAs, loaded on the argonaute protein 2 (Ago-2) bind to the mature mRNA and initiate the recruitment of the RISC complex triggering the degradation of the mRNA, dramatically scaling down gene expression. In vitro validation of gene silencing is a time expensive process and computational resources provide faster and cheaper options. Here, we identified the miRNAs target sequences on the mature PARP1 mRNA construct from the TargetScan database and obtained their complementary miRNA sequences from the miRbase. Using the human Ago-2 crystal structure deposited to the PDB database (PDB ID: 4W5Q) as a template, mutational studies were conducted on the protein/mRNA/miRNA complex to reproduce PARP1 mRNA seed pairing to miRNAs. Upon initial geometry optimization, further conformational search studies were done for thorough exploration of the conformational space of the protein/RNA complex by implementing conformational flooding (CF) molecular dynamics (MD) simulations. CF MD introduces a flooding potential to the force field, thus lowering energy barriers and facilitating observations of macroscale conformational changes. Clustering of the CF trajectories reveal the stable conformations of the Ago-2/miRNA/mRNA complexes. Energetically favorable miRNAs discovered in silico will further be validated with in vitro silencing assays.

Interrogation of Functional miRNA-Target Interactions by CRISPR/Cas9 Genome Engineering.

Posttranscriptional silencing by microRNAs (miRNAs) is a critical constituent of eukaryotic gene regulation. miRNAs are short (~22nt) noncoding RNAs capable of specifically targeting the miRNA-induced silencing complex (miRISC) to transcripts bearing a complementary miRNA response element (MRE). Although recent methodological advances have greatly improved our understanding of miRNA biogenesis and the mechanisms by which miRNAs repress their cognate targets, exploring the physiological relevance of direct miRNA-target interactions in vivo has remained an outstanding challenge. Here we describe the experimental protocol underlying a novel approach, which allows direct in situ interrogation of specific miRNA-MRE interactions by CRISPR/Cas9-mediated genome engineering (Bassett G et al., Nat Commun 5, 4640, 2014). In this instance, the CRISPR/Cas9 system is first used to catalyze homology-directed replacement of candidate MREs with molecular barcodes at endogenous loci. Subsequently, the effect of MRE mutation on transcript abundance (i.e., MRE activity) can be rapidly evaluated by routine quantitative PCR. This strategy enables functional investigation of a putative miRNA-target pair in a pool of transiently transfected cells, obviating the need for generation of clonal cell lines or transgenic animals. This protocol can be implemented in any cell line in less than 2 weeks and can readily be scaled up for multiplex studies. To facilitate the conceptual workflow underlying this strategy, we also describe a genome-wide resource for automated design and computational evaluation of CRISPR/Cas9 guide RNAs targeting all predicted MREs in various species (miR-CRISPR).

Metal-organic frameworks-based biosensor for microRNA detection in prostate cancer cell lines.

In this research, a novel dye-labeled probe (FAM-Probe) based on a nano metal-organic framework (NMOF) functionalized with folate (NMOF-FA) was prepared and applied as a fluorescent sensing platform for the recognition of intracellular microRNA (miRNA-21) in DU145, PC3, and LNCaP cancer cells. The NMOF-FA can be easily assembled with a dye-labeled miR-21 probe (FAM-Probe21), causing an efficient fluorescence quenching of fluorescence of FAM fluorophore. The probe can be specifically catch up by cancerous cells through targeting their folate receptor by folic acid on the FAM-Probe21-NMOF-FA complex. Upon the interaction of the FAM-Probe21-NMOF-FA with complementary miRNA (miR-21), the fluorescence intensity can be recovered, providing a specific system to detect miRNAs in prostate cancer cells. We used the proposed probe for cell-specific intracellular miRNA-21 sensing, following the alteration expression level of miRNA-21 inside living cells. Thus, the FAM-Probe21-NMOF-FA complex can be used as a new miRNA sensing method in biomedicine studies.

Development of a miRNA Sensor by an Inducible CRISPR-Cas9 Construct in Ciona Embryogenesis.

MicroRNAs (miRNAs) regulate multicellular processes and diverse signaling pathways in organisms. The detection of the spatiotemporal expression of miRNA in vivo is crucial for uncovering the function of miRNA. However, most of the current detecting techniques cannot reflect the dynamics of miRNA sensitively in vivo. Here, we constructed a miRNA-induced CRISPR-Cas9 platform (MICR) used in marine chordate Ciona. The key component of MICR is a pre-single guide RNA (sgRNA) flanked by miRNA-binding sites that can be released by RNA-induced silencing complex (RISC) cleavage to form functional sgRNA in the presence of complementary miRNA. By using the miRNA-inducible CRISPR-on system (MICR-ON), we successfully detected the dynamic expression of a miRNA csa-miR-4018a during development of Ciona embryo. The detected patterns were validated to be consistent with the results by in situ hybridization. It is worth noting that the expression of csa-miR-4018a was examined by MICR-ON to be present in additional tissues, where no obvious signaling was detected by in situ hybridization, suggesting that the MICR-ON might be a more sensitive approach to detect miRNA signal in living animal. Thus, MICR-ON was demonstrated to be a sensitive and highly efficient approach for monitoring the dynamics of expression of miRNA in vivo and will facilitate the exploration of miRNA functions in biological systems.

Femtomolar determination of an ovarian cancer biomarker (miR-200a) in blood plasma using a label free electrochemical biosensor based on L-cysteine functionalized ZnS quantum dots.

In the present study, a label-free electrochemical genosensor was designed based on ZnS quantum dots functionalized with l-cysteine (Cys-ZnS-QDs) to detect miR-200a, as a special ovarian cancer biomarker. The Cys-ZnS-QD genosensor was characterized by transmission electron microscopy (TEM), UV-Vis absorption and fluorescence methods. Cys-ZnS-QDs are electrodeposited on the glassy carbon electrode surface and act as a suitable substrate for immobilization of the DNA probe. The effective parameters in the preparation of the genosensor are optimized to improve its analytical performance. The analytical performance of the genosensor has been investigated using electrochemical impedance spectroscopy. Under optimal conditions, the linear range and the detection limit of miR-200a were found to be 1.0 × 10-14 to 1.0 × 10-6 M and 8.4 fM. In addition, the genosensor is used to detect the target complementary miRNA strand from a single-base mismatch miRNA strand. Finally, this label-free electrochemical biosensor was used to detect miR-200a in human plasma without using any amplification method.

Electrochemical biosensor for miRNA-21 based on gold-platinum bimetallic nanoparticles coated 3-aminopropyltriethoxy silane

We report an electrochemical biosensor based on gold platinum bimetallic nanoparticles (AuPtBNPs)/3-aminopropyltriethoxy silane (APTS) nanocomposite coated fluorine-doped tin oxide (FTO) as a biosensing platform for hybridization-based detection of miRNA-21. Field Emission-Scanning Electron Microscopy (FE-SEM), Fourier Transform Infrared Spectroscopy (FT-IR) and electrochemical measurements were carried out to ensure the successful construction of the biosensor. The amount of cDNA immobilized on electrode surface and hybridization time required for the miRNA-21 sensing were optimized. The biosensing platform showed detection limit of 0.63 fM with wide linear range i.e. 1 fM-100 nM for miRNA-21 detection. The biosensing strategy demonstrates a good recovery yield from 90.18% to 94.6% in serum samples. It offers good selectivity for its complementary miRNA compared to the non-complementary miRNAs. Other analytical features of the biosensor such as stability, reusability and reproducibility were also tested, providing appropriate results.

Molybdenum disulfide nanomaterial sensor-based detection of breast cancer microRNA

In this study, we sought to establish a rapid, simple, highly sensitive, and highly specific fluorescence sensor for the detection of breast cancer-related microRNA (miRNA) based on the novel nanomaterial known as molybdenum disulfide (MoS2). MoS2, a novel nanomaterial, was added to a fluorescently-labeled DNA probe system to construct a fluorescence sensor. Furthermore, non-complementary miRNAs, single-base mismatched miRNAs, and different concentrations of complementary miRNAs were hybridized with the fluorescently-labeled DNA probe. Then, miRNAs were detected by measuring changes in the fluorescence signals before and after hybridization. The detection was completed within 40 min. The detection limit of complementary miRNA reached pmol/L levels while distinguishing between complementary, non-complementary, and single-base mismatched miRNAs. The MoS2 fluorescence sensor demonstrated rapid analysis, high sensitivity, and high specificity. Additionally, this established detection method has the potential for application in the rapid detection of breast cancer-related miRNA as a promising cancer detection method.

Genome-wide identification of drought-responsive miRNAs in grass pea (Lathyrus sativus L.)

The grass pea (Lathyrus sativus L.) is an attractive legume which grows traditionally in drought-stricken, rain-fed and poor fertile soils. Globally drought is a major constraint for agriculture. Regulatory mechanism such as non-coding RNA is an important molecule to control gene expression in every domain of life. Therefore, identification and characterization of drought-responsive miRNAs of this plant can be very useful genomic resource to understand the molecular mechanisms of drought stress tolerance in plants. In this study we identified 143 and 128 conserved miRNAs by sequencing small RNA libraries from control (LC) and moisture-stressed (LD) leaf tissues. Additionally, 5 and 11 novel miRNAs were also found in LC and LD library respectively, among which 2 miRNAs were common. Further, we observed that 8 and 12 known miRNAs were up- and down- regulated under drought stress. Importantly, complementary miRNA (feeder) stands were also found for all the mature miRNAs. Expressions of few known and novel miRNAs were validated by qPCR in the tolerant genotype of grass pea under drought stress. The predicted target sequence of differentially expressed miRNAs were found to be either transcription factors such as F-box, U-Box or protein coding genes involved in proline, betain and osmolyte biosynthesis pathway indicating that these miRNAs may have specific role in imparting drought tolerance. These findings provide valuable information to understand the function of miRNAs in an inherently drought tolerant plant.

Preparation and application of AgNPs/SWCNTs nanohybrid as an electroactive label for sensitive detection of miRNA related to lung cancer

An electrochemical nanogenosensor based on silver nanoparticles/single-walled carbon nanotubes nanohybrid (AgNPs/SWCNTs nanohybrid), as a label, was developed for the analysis of miRNA (miR-25) related to lung cancer. The preparation steps of the proposed nanogenosensor were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The two components of the AgNPs/SWCNTs nanohybrid have different roles in the nanogenosensor performance. SWCNTs are responsible for interaction with the probe, while AgNPs produce electroanalytical signals. It was shown that the π-π interaction between the AgNPs/SWCNTs nanohybrid and the nitrogenous bases related to double-stranded nucleic acid is weaker than that for single-stranded nucleic acid. The different abilities of AgNPs/SWCNTs nanohybrid to bind with the probe hybridized with complementary (miRNA related to lung cancer), non-complementary and one-base mismatch targets led to identification of the nature and the concentration of miRNA through a simple and fast approach. The developed nanogenosensor was used to determine miR-25 in two concentration ranges of 1.0 × 10−12 to 1.0 × 10−10 M and 1.0 × 10−10 to 1.0 × 10−8 M with a limit of detection (LOD) of 3.13 × 10−13 M without PCR amplification. Furthermore, the nanogenosensor was employed to detect miR-25 in human plasma. This electrochemical procedure can be valuable in genetic analysis and early clinical diagnosis.

Hi-fidelity discrimination of isomiRs using G-quadruplex gatekeepers.

Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DNA probe-miRNA target hybridization is the primary, or initial, mode of detection. These errors then become exacerbated through subsequent amplification steps. Here, we present the design of DNA probes modified with poly-guanine (PG) tracts that were induced to form G-quadruplexes (G4) for hi-fidelity discrimination of miRNA core target sequence from single-base mismatched isomiRs. We demonstrate that, when compared to unmodified probes, this G4 'gate-keeping' function within the G4-modified probes enables more stringent hybridization of complementary core miRNA target transcripts while limiting non-specific hybridizations. This increased discriminatory power of the G4-modified probes over unmodified probes is maintained even after further reverse transcriptase extension of probe-target hybrids. Enzymatic extension also enhanced the clarity and sensitivity of readouts and allows different isomiRs to be distinguished from one another via the relative positions of the mismatches.

CRISPR-Cas9 Based Genome Editing Reveals New Insights into MicroRNA Function and Regulation in Rice.

MicroRNAs (miRNAs) are small non-coding RNAs that play important roles in plant development and stress responses. Loss-of-function analysis of miRNA genes has been traditionally challenging due to lack of appropriate knockout tools. In this study, single miRNA genes (OsMIR408 and OsMIR528) and miRNA gene families (miR815a/b/c and miR820a/b/c) in rice were targeted by CRISPR-Cas9. We showed single strand conformation polymorphism (SSCP) is a more reliable method than restriction fragment length polymorphism (RFLP) for identifying CRISPR-Cas9 generated mutants. Frequencies of targeted mutagenesis among regenerated T0 lines ranged from 48 to 89% at all tested miRNA target sites. In the case of miRNA528, three independent guide RNAs (gRNAs) all generated biallelic mutations among confirmed mutant lines. When targeted by two gRNAs, miRNA genes were readily to be deleted at a frequency up to 60% in T0 rice lines. Thus, we demonstrate CRISPR-Cas9 is an effective tool for knocking out plant miRNAs. Single-base pair (bp) insertion/deletion mutations (indels) in mature miRNA regions can lead to the generation of functionally redundant miRNAs. Large deletions at either the mature miRNA or the complementary miRNA* were found to readily abolish miRNA function. Utilizing mutants of OsMIR408 and OsMIR528, we find that knocking out a single miRNA can result in expression profile changes of many other seemingly unrelated miRNAs. In a case study on OsMIR528, we reveal it is a positive regulator in salt stress. Our work not only provides empirical guidelines on targeting miRNAs with CRISPR-Cas9, but also brings new insights into miRNA function and complex cross-regulation in rice.

A metal-organic framework based PCR-free biosensor for the detection of gastric cancer associated microRNAs

We report herein five sensing platforms for the detection of five gastric cancer associated microRNAs (miRNAs). The sensing platforms are hybrids formed from a water-stable metal organic framework (MOF) {[Cu(dcbb)2(H2O)2]·10H2O}n (1, H2dcbbBr=1-(3,5-dicarboxybenzyl)-4,4′-bipyridinium bromide), respectively with five carboxyfluorescein (FAM) labeled probe single-stranded DNA (probe ss-DNA, denoted as P-DNA). Within the hybrid, MOF 1 tightly interacts with the P-DNA through electrostatic and/or π-stacking interactions and results in fluorescence quenching of FAM via a photo-induced electron transfer (PET) process. In the presence of the complementary target miRNAs miR-185, miR-20a, miR-92b, miR-25 and miR-210, which are expressed abnormally in the plasma of gastric carcinoma patients, P-DNA is released from the surface of MOF 1 ascribed to the stronger base pair matching, leading to the FAM fluorescence recovery. Each P-DNA@1 system is effective and reliable for the detection of its complementary target miRNA with the detection limits from 91 to 559pM, and is not interfered by other four miRNA sequences.

Transcriptomes of Podophyllum hexandrum unravel candidate miRNAs and their association with the biosynthesis of secondary metabolites

Podophyllotoxin is a high value metabolite having anticancerous activity and procured from a medicinal plant Podophyllum hexandrum. The escalating demand for podophyllotoxin necessitates development of alternate production platforms. Cell cultures have been tried in the past, however, podophyllotoxin yields were very low (0.3%). Multiple genes of biosynthetic pathway have been correlated with podophyllotoxin content. Understanding regulation of podophyllotoxin is, therefore, essential to design a suitable genetic intervention strategy. Six potential miRNAs were identified in NGS transcriptomes of P. hexandrum for which eight potential mRNA targets were discerned. The identified miRNAs and their mRNA targets (UDP glycosyltransferase, flavonol synthase, glyceraldehyde 3-phosphate dehydrogenase, peroxidase, malate dehydrogenase, phosphoenolpyruvate carboxylase, WRKY 37 and MYBF1 transcription factors) were further tested for any correlation through qRT-PCR. miR396b, miR2673a, miR828b and miR2910 established negative correlation with their mRNA targets through downregulation via complementary miRNA:mRNA base pairing. This is the first report on identification, characterisation, validation and expression analysis of miRNAs and their targets in P. hexandrum.

An interference-free and label-free sandwich-type magnetic silicon microsphere -rGO-based probe for fluorescence detection of microRNA

An interference-free and label-free sensing platform was developed for the highly sensitive detection of microRNA-21 (miRNA-21) in vitro by magnetic silicon microsphere (MNP)-reduced graphene oxide (rGO)-based sandwich probe. In this method, DNA capture probes (P1) were connected with MNPs at the 5′ end and hybridized with completely complementary target miRNA. Subsequently, rGO was retained and induced the fluorescence quenching in the supernatant. Through the magnetic separation, the supernatant environment was simplified and the interference to analytical signal was eliminated. When DNA capture probe-modified magnetic silicon microspheres (MNP-P1) were adsorbed through rGO in the absence of a target and formed a sandwich structure, the formed nanostructure was easily removed from the solution by a magnetic field and the fluorescence intensity was maximally recovered. This proposed strategy, which both overcame the expensive and cumbersome fluorescent labeling, and eliminated interference to analytical signal for guaranteeing high signal-to-background ratio, exhibited high sensitivity with a detection limit as low as 0.098nM and special selectivity toward miRNA-21. The method was potentially applicable for not only detection of miRNA-21 but also various biomarker analyses just by changing capture probes.

Ultra-sensitive fluorescent sensor for intracellular miRNA based on enzyme-free signal amplification with carbon nitride nanosheet as a carrier.

A novel ultra-sensitive fluorescent sensor for monitoring microRNA (miRNA) in living cells was constructed by utilizing a hybridization chain reaction (HCR) as the signal amplification with a carbon nitride nanosheet (CNNS) as a carrier. The Cy5-labeled hairpin DNA could be adsorbed onto the surface of CNNS, resulting in fluorescence quenching of Cy5. When treated with complementary miRNA, the fluorescence was recovered because miRNA could efficiently trigger an HCR, which led to the release of the HCR products from the CNNS. This intracellular HCR strategy can be used for ultra-sensitive monitoring of intracellular miRNA. The main advantages of the proposed method are its simplicity, high sensitivity, high specificity and low toxicity for monitoring low-level biomarkers.

Identification of circular RNAs as a promising new class of diagnostic biomarkers for human breast cancer.

Endogenous noncoding circular RNAs (circRNAs) have gained attention for their involvement in carcinogenesis, but their expression pattern in breast cancer has remained largely unknown. In this two-stage study, we first used an Arraystar Human circRNA Array to construct a genome-wide circRNA profile. We then selected candidate circRNAs for validation using a quantitative real-time polymerase chain reaction system. CircRNA/miRNA interactions were predicted and sequence analyses were performed. Among 1155 differentially expressed circRNAs, 715 were upregulated and 440 were downregulated in breast cancer tissues. The validation study demonstrated that hsa_circ_103110, hsa_circ_104689 and hsa_circ_104821 levels were elevated in breast cancer tissues, whereas hsa_circ_006054, hsa_circ_100219 and hsa_circ_406697 were downregulated. These circRNAs targeted complementary miRNA response elements. The area under the receiver operating characteristic curve for distinguishing breast cancer was 0.82 (95% CI: 0.73-0.90) when hsa_circ_006054, hsa_circ_100219 and hsa_circ_406697 were used in combination. This study provides evidence that circRNAs are differentially expressed in breast cancer and are important in carcinogenesis because they participate in cancer-related pathways and sequester miRNAs.

Triangular silver nanoplates: Properties and ultrasensitive detection of miRNA

Triangular silver nanoplates (TSNPs) have been functionalised with probe strand miRNA that is complementary in part to the target nucleic acid, miR-132-3p, that is associated with neuroblastoma. These TSNPs were immobilised in gold microcavities via complementary miRNA hybridisation and can give plasmonic enhancement of the Raman response. Optimum enhancement can be achieved by using different excitation wavelengths and changing the distance between the nanoplate and the surface of the cavity. As silver is electrocatalytically active to the reduction of hydrogen peroxide, these probe-functionalised TSNPs can be used in a miRNA assay where a linear response was obtained for target concentrations from 100fM to 1μM target concentration, with a current generated of 100μA.

Effects of exposure to Streptococcus iniae on microRNA expression in the head kidney of genetically improved farmed tilapia (Oreochromis niloticus)

BackgroundGenetically improved farmed tilapia (GIFT, Oreochromis niloticus) are susceptible to infection by Streptococcus iniae when maintained in modern intensive culture systems. GIFT are commercially important fishes that are cultured widely in southern China. The role of microRNAs (miRNAs) in the regulatory response of GIFT to S. iniae infection has been underestimated and has not yet been well studied. Head kidney has an important immune function in teleost fishes. The main aim of this study was to determine the possible function of miRNAs in head kidney of S. iniae-infected GIFT. MiRNAs are small, non-coding RNAs that regulate gene expression by binding to the 3’-untranslated regions of their target mRNAs. MiRNAs are known to regulate immune-regulated signaling and inflammatory response pathways.ResultsHigh-throughput deep sequencing of two libraries (control group [CO] and infected group [IN]) of RNA extracted from GIFT head kidney tissues generated 12,089,630 (CO) and 12,624,975 (IN) clean reads. Bioinformatics analysis identified 1736 and 1729 conserved miRNAs and 164 and 165 novel miRNAs in the CO and IN libraries, respectively. Three miRNAs (miR-310-3p, miR-92, and miR-127) were found to be up-regulated and four miRNAs (miR-92d-3p, miR-375-5p, miR-146-3p, and miR-694) were found to be down-regulated in the S. iniae-infected GIFT. The expressions of these miRNAs were verified by quantitative real-time PCR. RNAhybrid and TargetScan were used to identify complementary miRNA and mRNA target sites, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used to annotate and predict potential downstream regulation of biological pathways. Seven target genes, which encode immune-related proteins (complement C3, cytidine deaminase, regulator of G-protein Rgs22, mitogen-activated protein kinase Mapk1, metabotropic glutamate receptorm GluR8, calcium-sensing receptor CaSR, and microtubule-associated protein Map1S) were predicted to play crucial roles in the GIFT response to S. iniae infection.ConclusionsS. iniae outbreaks have hindered the development of the tilapia industry in China. Understanding the miRNA transcriptome of S. iniae-infected GIFT is important for exploring the immune responses regulated by miRNAs as well as for studying novel regulated networks to prevent and treat S. iniae infections in the future.

Micro-RNA detection based on fluorescence resonance energy transfer of DNA-carbon quantum dots probes

Carbon quantum dots have been proposed as an effective platform for miRNA detection. Carbon dots were synthesized by citric acid. The synthesized dots were characterized by dynamic light scattering, UV-Vis spectrophotometry, spectrofluorimetry, transmission electron microscopy and FT-IR spectrophotometry. The fluorescence quantum yield of the synthesized dots was determined using quinine sulfate as the standard. The FAM-labeled single stranded DNA, as sensing element, was adsorbed on dots by π-π interaction. The quenching of the dots fluorescence due to fluorescence resonance energy transfer (FRET) was used for mir 9-1 detection. In the presence of the complementary miRNA, the FRET did not take place and the fluorescence was recovered.