At the same time, with the development and progress of sequencing technology, some methods of nucleic acid extraction and library preparation have been derived. For example, it has been successfully harnessed from single cellsFor RNA and DNA library preparation. NGS library preparation is based on modifying the targeted nucleic acid, RNA, or DNA into a form that can be used by the sequencer (Fig. 1). Here, we compare multiple library preparation strategies and NGS applications, focusing on libraries that are compatible with illumina sequencing technology.
Principle The principle of second-generation sequencing is library construction, and the fragment generated by PCR or the fragment interrupted by the genome needs to add joint modification at both ends to be sequenced. End modification. The broken fragments are randomly broken, and their ends may be uneven. Therefore,The first step in building a warehouse is to fill out the uneven ends. Add a connector.
Introduction: Since the Roche 454 sequencer led the way in 2005, second-generation sequencing (NGS) technology has made a revolutionary leap forward, and the popularity of Illumina has lowered the bar.
Library construction: Careful design of terminal modification and connector addition, the use of NEBNext and other kits, to ensure the specificity of the library and sequencing compatibility. Magnetic bead purification is like the process of purifying the soul, removing impurities and ensuring the purity of the data. For the Illumina platform, the sequencing process is unique: temperature, enzyme and fluorescenceThe precise control of the light signal is like a conductor directing the performance of each DNA sequence.
Library Construction begins with the preparation of genomic DNA (although sequencing companies require a sample size of 200ng, the Gnome Analyzer system requires a sample size as low as 100ng, which can be used in many experiments with limited samples). The DNA is then randomly fragmented into small fragments of a few hundred bases or less, with specific adaptors at each end.
In summary, antisense long non-coding Rnas have demonstrated their important role in gene expression regulation through fine regulation with justice chain genes. These findings not only broaden our understanding of lncRNA function, but also provide new strategies for fighting disease. The mystery of antisense long non-coding RNA is being uncovered layer by layer, waiting for our further exploration and application.
Exploring the Secret Code of Life: The Mysteries of Long non-coding Rnas in the eukaryotesLong non-coding Rnas (lncrnas), which are more than 200 nucleotides long, are a mysterious molecular guardian in the genetic code of living things but do not code for proteins.
Non-coding RNA (ncRNA) is a kind of mysterious molecules in the body, they do not encode proteins, but play a role in the RNA level. These Rnas are so diverse that they can be divided into distinct classes based on length and function, and their discovery reveals a whole new dimension of genomic expression.
At the RNA levelThey can perform their biological functions. Non-coding Rnas can be divided into three categories in terms of length: less than 50 nt, including microRNA, siRNA, piRNA; 50 nt to 500 nt, including rRNA, tRNA, snRNA, snoRNA, SLRNA, SRPRNA, etc.; Greater than 500 nt, including long mRNA-like non-coding Rnas, long non-coding Rnas without polyA tails, etc.
Long non-coding Rnas in gene clusters and even throughout chromosomesHorizontal cis-regulatory effect. The regulation of "dose compensation" in drosophila is roX RNA, which also has a trans-regulatory role and together with other proteins forms the MSL complex that regulates X chromosome activity in male drosophila. Xist RNA regulates the inactivation of X chromosome in mammals, and it has a special mode that can interact with some proteins to achieve the inactivation of X chromosome.
mirnas are a class of endogenous non-coding Rnas in eukaryotes that regulate gene expression, with a length of 20-25nt (note the difference between NT and bp here)I have always been confused before, bp is a base pair, a pair of bases, mostly found in DNA; nt is short for nucleotide, and for single-stranded RNA, it is described by nt).
Use Antisense relation and Cis relation and Trans relation. lncRNA has many ways of regulation, which not only has a regulatory effect on mRNA, but also canIt acts as a precursor of miRNA in vivo. Figure 1 shows several regulatory modes of lncRNA.
lncRNA regulates mRNA gene expression, but the regulated mRNA gene should be far away from the lncRNA coding site (taking into account the distance factor), which may also change the spatial structure of the remote mRNA gene, thus affecting the expression of the mRNA.
A Whole transcriptome sequencing detects small RNA, circRNA, lncRNA and mRNA, aiming to analyze their interaction network relationships in cells and detect themThe solution is to build two libraries (one with 10M small RNA data and the other with 12G lncRNA circRNA mRNA data), which requires a larger sample size than transcriptome sequencing.
Generally speaking, lncrnas regulate gene expression mainly from three aspects: epigenetics, transcriptional regulation and post-transcriptional regulation. 1. Epigenetic regulation Studies on lncrNA-mediated epigenetic changes in mammals originated from genomic printing and X chromosome inactivation (X chrom)osome inactive) were closely related to H19 and X ist RNA, respectively.
lncRNA, full name of "long non-coding RNA", is a hot topic in RNA research in recent years. In the past, I only heard about various lncRNA sequencing, but did not seriously do the analysis. Recently I received a new task, so I came to learn more about it. I am really forgetful, so I wrote down the basic information of lncRNA.
lncRNA sequencing is used to study existing reference genome speciesThe positioning of tissue cells is very accurate, and paraffin section is more suitable for the long-term preservation of specimens. I will arrange some for your reference, embedding, and detecting the primary antibodies of such antigens can only be frozen section, because the detected antigens are easy to be destroyed and cause the antigen to disperse, which is more complicated and wax dipping.
3. For differentially expressed lncrnas, the upstream and downstream sequences of transcription start sites were extracted, and their transcription factor binding sites were predicted by using prediction programs. Currently, lncRNA chips on the market all contain mRNA expression probes, and the analysis results of lncRNA target genes are combined with the chipsCorrelation analysis of mRNA expression results can further analyze the function of lncRNA.
1. The full transcriptome is not the full-length transcriptome, which refers to the detection of ordinary mRNA, plus three conventional non-coding genes such as lncRNA, miRNA and CircRNA, while the full-length transcriptome refers to the adoption of third-generation sequencing technology when sequencing, which can base the entire length of the gene transcript productThe base is sequenced at one time, which makes it easy to know the difference between different variable cut transcripts.
2. The total transcriptome is a high-throughput technique that can study all RNA molecules in an organism or tissue. It is a comprehensive sequencing of RNA molecules and is a very powerful bioinformatics research method. Through the comprehensive transcriptome, information such as the quantity, type and expression level of RNA in organisms or tissues can be comprehensively explored, which provides important support for our biological research.
3. Transcriptomic analysis mainly studies the gene expression of organisms in different physiological states. tongHigh-throughput sequencing technology can obtain the sequence and abundance information of biological transcripts, so as to analyze the spatio-temporal specificity, regulatory mechanism and function of gene expression. For example, in cancer research, scientists have found many abnormal gene expression related to the occurrence and development of cancer through transcriptomic analysis, providing new ideas for the diagnosis and treatment of cancer.
4, transcriptome sequencing is a high-throughput sequencing technology that can comprehensively analyze and identify the type, amount, and sequence of RNA in a single cell or tissue. This method can reveal information about gene expression, gene regulation, shear variation, etc., fromIt helps us understand the biological functions of cells and tissues more deeply.
5. Transcriptomics is the study of the comprehensiveness and dynamics of gene transcription processes. The application of bioinformatics in transcriptomics is mainly to reveal gene expression patterns, splicing isomers and regulation of transcription factors by analyzing RNA sequence data. RNA sequencing and analysis RNA sequencing (RNA-SEQ) is a high-throughput method that can detect and quantify all RNA molecules within cells, including mRNA, miRNA, and lncRNA.