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Original link:Metaorganisms - circRNA sequencing
brief introduction
Covalent closed, single stranded circular RNA (circRNA) is a relatively special type of RNA that does not have a free 5 'cap structure or a 3' poly (A) structure, and is not sensitive to nucleases, making it more stable than regular linear RNA. With the rapid development of high-throughput sequencing and bioinformatics analysis technology, thousands of circRNAs have been identified in different species, most of which are conserved and stable between different species, and the expression of circRNAs is cell specific, tissue specific, and developmental stage specific. In addition to the conventional ceRNA regulatory mechanism that competes for miRNA binding, circRNA also has new functions such as translation peptides, which have great potential in medical and animal and plant basic research and later transformation.
Initial sample size and sample delivery recommendations
Sample type | initiation mass |
Animal and clinical organ tissues/brain tissues, etc | >20mg |
Animal and clinical skin/bone/blood vessels/adipose tissue, etc | >100mg |
Plant leaf tissue/flowers | >200mg |
Plant roots/stems/fruits/seeds | >500mg |
Primary cells/cell lines | >5 x 106 pieces |
Neutrophils/eosinophils/basophils | >5 x 107 pieces |
Extracellular vesicle samples | >1x108 pieces |
Serum/plasma/cerebrospinal fluid/joint effusion/follicular fluid | >2mL |
cell culture supernatant | >20mL |
urine | >30mL |
Total RNA | >1 μ g and RIN>7.0 |
matters needing attention:
① It is recommended to store the tissue samples in relevant tissue preservation solutions such as RNAlate, RNAOld, RNAProtect, etc., and then store them at -80 ℃ or send them on dry ice;
② After sufficient lysis using TRIzol or other lysis solutions, the cell samples are stored at -80 ℃ or sent on dry ice;
③ For more detailed sample preparation guidelines, please contact online customer service
Bioinformatics analysis process and content
circRNA | Analysis content | remarks |
Sequencing data quality control | Raw data quality control statistical table | |
Reference genome alignment analysis | Reference genome gene statistics table | |
Reference genome alignment reads statistics table | ||
Statistical Table of the Number of CircRNA Identification Samples | ||
Statistical chart of reference genome alignment region distribution | ||
Reference genome nonlinear alignment region distribution statistical chart | ||
Quantitative analysis of circRNA identification | CircRNA fitting sequence | |
CircRNA expression profile | ||
BSJ statistical table for circRNA | ||
CircRNA statistical analysis | CircRNA type statistical pie chart | |
Statistical boxplot of circRNA expression distribution | ||
Distribution map of circRNA expression density | ||
Statistical chart of the distribution of circRNA chromosome numbers | ||
Statistical chart of the number of circRNA exons | ||
Statistical chart of the number of circRNAs in parental genes | ||
CircRNA differential analysis | Statistical chart of the number of differential circRNAs | |
Analysis of differential expression of circRNA in CaseVSControl comparison group | Case VSControl comparison group circRNA volcano map | |
Case VSControl comparison group circRNA heatmap | ||
CaseVSControl comparison group circRNA scatter plot |
Application scenarios and cases
Application scenario 1: Structural molecule recruitment of core proteins and nucleic acids
Applicable scope: Any research direction such as basic medicine, biochemistry, and molecular biology
LncRNA and circRNA sometimes act as special RNA molecules, forming protein protein complexes or backbone molecules of protein nucleic acids, exerting a series of regulatory effects on downstream molecules. Some circRNAs recruit ubiquitinases to degrade downstream proteins, or some lncRNAs recruit transcription factors to bind to the promoter region of the target gene to activate downstream gene expression.
Application scenario 2: Exosomes
Scope of application: Any research direction in clinical and translational medicine, basic medicine, etc
Due to the fragmented form of lncRNA in exosomes, this random fragment is not only difficult to detect, but also has lower concentrations and poor repeatability, making lncRNA not one of the hot molecules in exosome research. However, due to its stability, circRNA can not only be isolated from extracellular vesicles in body fluid samples as a disease marker, but also be used for later treatment of extracellular vesicles, making it one of the more popular sub research directions for circRNA in the post ceRNA era. One direction is disease biomarkers, which are generally studied in large sample cohorts. One is the research direction of late stage transformation and mechanism, including the molecular mechanism of extracellular vesicle receptor cells and disease treatment.
Application scenario 3: Translating peptides
Scope of application: Any research direction including clinical and translational medicine, basic medicine, biochemistry and molecular biology, etc
There is an untranslated RNA sequence on circRNA, called IRES, which can fold into a structure similar to the starting tRNA and recruit more ribosomes. Under normal circumstances, IRES does not bind to eIF, but rather to a class of proteins called ITAF. The function of ITAF is to recruit ribosomes into the internal structure of circRNA to initiate protein translation. In addition, there may also be IRES enhancers, similar to UTR elements of circ-ZNF609. Some studies have also found through transcriptome sequencing and transcriptome sequencing that non coding RNA LINC-PINT can be translated into functional peptides after forming circular RNA.
Application scenario 4: ceRNA regulatory mechanism
The full name of ceRNA is competitive endogenous RNA, which is an element that can compete for binding to RNA. Usually, lncRNA and circRNA compete to bind to miRNA, and we generally refer to lncRNA and circRNA as ceRNA. The full name of ceRNA regulation network is ceRNA regulation network, which refers to the entire regulatory network cascade in which ceRNA participates. And ceRNA analysis refers to the analysis of the entire ceRNA regulatory network. Generally, there are circRNA miRNA mRNA analysis or lncRNA miRNA mRNA analysis.
Project Process Project Process
Transcriptome sequencing with eukaryotic involvement in metaorganisms can provide researchers with a complete range of service processes, including sample extraction, library sequencing, and data analysis, providing high-quality data results and providing strong reference for subsequent research.
