miRNA functional analysis strategy
Abstract: In different stages of development or differentiation and disease models, using miRNA-specific chips can map the miRNA expression profiles of cells and tissues, which can reveal the specific miRNAs involved in these processes. Overexpression and silencing of miRNA is a powerful method to study the function of miRNA. These studies should be performed in cells or in vivo and combined with phenotype and gene expression analysis.
Lin-4, the first known miRNA, was discovered in 1993. Since then, miRNA research has been out of control. Studies on nematodes, fruit flies and other model organisms have identified many important functions of miRNA, including coordination of cell proliferation and death during development, stress resistance, fat metabolism, and so on. Compared to these lower model organisms, mammalian miRNA function research can be much more complicated.
In the first few years, scientists ’primary goal was to use cloning, bioinformatics, and gene expression to compile a complete miRNA catalog and its expression. As this goal approaches, the focus of research shifts to the interpretation of miRNA function. In response to this goal, a variety of techniques have emerged, including reporter gene analysis, in situ hybridization, overexpression and silencing, bioinformatics prediction algorithms, etc., which all contribute to the inference of miRNA function.
Analysis of miRNA expression
When identifying new miRNAs, three methods are usually used: forward genetics, directional cloning and bioinformatics analysis. Forward genetics is mainly used in fruit flies and nematodes, and it uses a mutant phenotype to understand the function of miRNA. This is how the first miRNA gene lin-4 was identified. However, forward genetics has identified few miRNAs over the years. This is because miRNAs are so small that as long as mutations do not affect the seed sequence, they have the potential to tolerate, so it is difficult to hit. In addition, due to redundancy, phenotypic screening cannot recognize many miRNA mutants. Therefore, forward genetics cannot be the main force for identifying miRNA.
Later, the emergence of directional cloning technology enabled the large-scale identification of miRNAs in various cell lines and tissues, including humans, mice, and zebrafish. The miRNA cloning procedure is roughly as follows: RNA samples are separated on a denatured polyacrylamide gel, and fragments with a size of 18-25 nt are recovered. Next, add 5 'and 3' linkers to the RNA, and perform RT-PCR, then clone the fragment into a vector to construct a cDNA library. Single clones were sequenced and analyzed to determine small RNA. The current rapid development of sequencing technology has greatly promoted this method. However, it is difficult to identify certain miRNAs that are expressed only in specific cell types or expressed at low levels. In addition, due to physical properties or post-transcriptional modifications, some miRNAs may be difficult to clone, which is also a difficult problem.
At the same time, scientists have also developed a variety of algorithms to predict miRNA. All methods make use of secondary structure information, because the presence of hairpin structure is the main feature of miRNA. Many of these methods also rely on sequence conservation to distinguish miRNA candidates from unrelated genomic hairpins. Other methods evaluate the thermodynamic stability of the hairpin structure, which is similar to the sequence and structure of known miRNAs. Another efficient method is to explore the genomic sequence around known miRNAs, because many miRNAs are arranged in clusters. Many miRNAs in humans and mice were identified in this way. Of course, the candidate miRNA predicted by the computer still needs experimental verification.
Sometimes, we do not need to understand all miRNAs, but just want to understand the miRNAs that are specifically expressed in different stages of development or differentiation, and under certain disease states. At this time, the chip becomes a powerful tool that can monitor tissue-specific miRNA expression and help you pick out key miRNAs. Many miRNA chips currently on the market include the latest Sanger miRBase database version 14.0, allowing you to keep up with the situation. Although the initial cross-hybridization and specificity of miRNA chips were criticized, many have been able to distinguish single base differences after improvement. With it, people draw miRNA expression profiles during cell differentiation; with it, people learn about abnormal miRNA expression patterns in disease states. ABI has also developed a chip-like TaqMan miRNA Array, which gathers hundreds of TaqMan miRNA Assays on a microfluidic card. Although the throughput is not as high as the chip, it takes advantage of TaqMan analysis-high sensitivity, high Specificity and wide dynamic range.
Identify the function of miRNA
The functional study of miRNA is similar to genes, and overexpression and silencing are two powerful methods. The induced expression of miRNA is often the first step in many studies. Transfecting miRNA mimetics (sold by multiple companies) into cells can achieve transient overexpression of miRNA. But long-term research depends on plasmids. These recombinant plasmids continuously produce functional miRNAs, which is also very simple to design. Using common protein expression vectors, the miRNA sequence is cloned, and under the cleavage of Dicer enzyme, mature miRNA can be generated. Some research groups also introduced these plasmids into adenovirus or lentivirus systems to overcome the low transfection efficiency of primary cells or stem cells, or to introduce miRNA into mice. However, the conclusion that only miRNA overexpression is often unconvincing, we still need loss-of-function experiments to confirm.
For miRNA studies in mice, scientists have developed genetic methods to generate loss-of-function mutations. At present, there are three main types: (1) mutation of Dicer enzyme, so that all mature miRNAs are deleted; (2) knockout of miRNA gene in mice; (3) mutation of miRNA target site. However, the high degree of redundancy of miRNAs poses considerable challenges to this loss of function research. Moreover, many miRNAs are arranged in clusters. The deletion or interference of a miRNA may affect the correct folding and processing of the polycistronic transcript, thereby affecting the expression of adjacent miRNAs. In recent years, anti-sense targeted non-genetic methods have been used more frequently. Several companies provide miRNA inhibitors. These 2'-O-methyl modified oligonucleotides irreversibly inhibit the function of miRNA.
The above methods provide a framework for miRNA functional studies in vitro and in vivo. In different stages of development or differentiation and disease models, the use of miRNA-specific chips can map the miRNA expression profiles of cells and tissues, which can reveal the specific miRNAs involved in these processes. Overexpression and silencing of miRNA is a powerful method to study the function of miRNA. These studies should be performed in cells or in vivo and combined with phenotype and gene expression analysis.
Similar to transcription factors, miRNAs are a large class of gene regulatory molecules. Their unique mode of action requires new analytical strategies. Techniques for systematically identifying miRNAs and studying the biological effects of over-expression or silencing of specific miRNAs already exist, but it is also necessary to understand the signaling pathways that affect miRNA function, as well as the environmental or genetic factors that affect miRNA expression. Once this information is obtained, it is possible for researchers to design new treatment strategies to treat genetic and acquired diseases.
A pet harness is equipment consisting of straps of webbing that loop nearly around-that fasten together using side release buckles-the torso of an animal.
These harnesses generally are made to have both a strap on the chest in front of the forelimbs, and a strap around the torso behind the forelimbs, with straps in between connecting these two. Having a D-ring suitable for (pet tags and) a leash to clip to, they are most often used to restrain an animal, but dogs also particularly wear them to assist a person with a disability or haul people and items. There is also the lifting harness for dogs with disabilities, covered in this article.
Some come in different sizes, although many are size-adjustable with tri-glide slides to loosen or shorten the straps` length. The straps may come in a range of different colors, and some have reflective coating.
Pet Harness,Dog harness,Classic Dog Harness
Yangzhou Pet's Products CO.,LTD , https://www.yzpqpets.com