The Detection Methods Study Of MicroRNA

MicroRNA (miRNAs) is a class of protein non-coding, endogenous, small RNAs found in animals and plants. These highly conservative, 19-25 nucleotides (nt) long miRNAs are thought to regulate gene expression by binding to the 3'-untranslable regions (3'UTRs) of mRNAs. Promotion of the degradation of target mRNAs and suppression of protein translation are considered to be the main mechanisms of regulation of gene expression. However, more researches are needed to explore the precise mechanisms of miRNAs regulations. It is believed that miRNAs play a critical regulatory role in a wide range of cell functions e.g. metabolism, developmental process as well as many biological processes including cell apoptosis, DNA damage and tumorigenesis. Most importantly, changes of the expression level of miRNAs have been associated with many kinds of diseases, suggesting that miRNAs may have potential as diagnostic markers and targets for cancer therapies. For further research about the function of miRNAs, a method which can profile the miRNAs expression patterns from different tissues, organs and development stages sensitively and specifically is needed to be established.The intensive studies on differential expression patterns and regulatory roles of miRNAs call for sensitive and specific method to detect trace amount of these small size, high sequence homological miRNAs. Northern blot is the"gold standard"method for miRNAs quantification with its derivation assays, but these methods are time-consuming and require a relatively large amount of total RNA, which might limit their applications. Several comparatively simple methods, such as cDNA arrays, primer extension, stem-loop RT-PCR have also been developed. However, the common trait that these methods rely on expensive equipment and an advanced detection system limits their application in most general labs.We have developed a kind of miRNAs detection method based on the complementary binding of oligonucleotides probes and gold nanoparticle with silver enhancement. This assay is easy and rapid for miRNAs detection without complicated instruments. Therefore, it is suitable for cost-effective detection of miRNAs, which may establish a new way for future study of miRNAs. At the same time, we developed a novel and simple miRNAs detection system through self-primer amplification (SPA) using a template containing two miRNAs complement sequences with promoter sequence inserted. The detection limit of SPA assay is 100pg of total RNA with a wide range of lineage relationship, so this method is hoped to be implemented by common lab.PartⅠ: Application of gold nanoparticle technology in the detection of microRNAObjective: To explore the application of gold nanoparticle technology in detection of microRNA. Methods: Gold nanoparticle labeled thiol-modified oligonucleotide probes and miRNAs were incubated to form the hybridization double strands, then the biotin labeling probes were hybridized with miRNAs of double strands to form a complex. The complex was then immobilized on the surface of the microplate by the streptavidin embedded previously. The unbinding probes in the mixture were then washed away by 1×PBS and 2×PBN solution. At last, it was treated with Silver Enhancement solution and read by Microplate Reader at 630nm. As the target miRNAs binding with nanoparticle-modified probes, the amount of nanoparticle was proportional to the miRNAs, which were correspondingly related to the optical density. Results: The color change of the solution was obviously consistent with the concentration of miRNAs, while not observed in negative controls. At a controlled point of time, the concentration is higher, and the OD value is corresponding higher. In the range of 10pM to 10fM, the OD value of the solution induced by miRNAs was produced in a dose-dependent manner at the same reaction time. The dose-response relationship curve about miRNAs concentration against the OD value was set up (R2=0.9906). The minimum detection limit for miRNAs is 10fM and this method can differentiate the sequences with single nucleotide mismatch. Conclusion: The gold nanoparticle probes technology is a rapid, sensitive, specific, and cost-effective method to detect miRNAs,and is hoped to be applied to screen of miRNAs in biological samples.PartⅡ: A novel detection of microRNA by real-time self primer amplificationObjective: To develope a molecular biological method to detect miRNAs based on self primer amplification. Methods: Enzymes (DNA polymerase and RNA polymerase) and template oligonucleotides (about 60-nt) were used in self-primer amplification assay. This template oligonucleotides contained two pieces of target miRNAs'complement sequences at the two terminals each and a piece of promoter sequence in the middle of template. Target miRNAs binding with template complementarily to form imperfect double strands, at the catalysis of Taq DNA polymerase, the target miRNAs was extended to form perfect double strands. Then, a RNA which has the same sequence with miRNAs was generated by the RNA polymerase at the downstream of promoter, and these transcripts went into the next amplification as substrate. The cycle is an isothermal amplification process, and the amplification signal is detected by SBYR Green. Results: By this method, as few as 1fM of synthetic miRNAs and miR-122a/miR-128 in 100 pg total RNA samples from mouse liver and brain tissue could be quantified. The log values of target miRNAs and Ct values showed an obviously linearity relationship (R2=0.9848) with a dynamic range as wide as over 7 orders of magnitude. Conclusion:Although the throughput of SPA assay needs to be improved, this method enables fast, accurate and sensitive miRNAs profiling and can be used for the quantification of other small RNA molecules such as short interfering (SiRNAs).