Rna Ligation Efficiency
Rna Ligation Efficiency - Related Videos
14:15
Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing
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Cited by 6 •
2014
MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.
07:34
DNA Ligation Reactions
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2023
In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. This video provides an...
11:00
A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium
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Cited by 83 •
2010
This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.
11:32
Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen
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Cited by 20 •
2017
Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.
11:53
Cecal Ligation Puncture Procedure
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Cited by 200 •
2011
The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.
FAQs
Many RNA ligases join an RNA 5′ phosphate to a 3′ hydroxyl through enzyme-mediated activation steps. Efficiency therefore depends not only on whether the enzyme is present, but also on whether the substrate ends can participate in these steps. Differences in end chemistry can change how readily molecules are joined and can influence the completeness of an RNA manipulation.
RNA ligation efficiency varies with substrate-end chemistry, nucleotide sequence, and molecular structure. These features can affect enzyme access, activation, or alignment of the reacting ends. As a result, two RNA molecules exposed to the same ligase may not join equally well, creating unequal representation when ligation is used to prepare or analyze RNA samples.
Sequence and structure can make some RNA molecules better substrates than others, producing ligation bias. This bias changes the relative representation of transcripts after joining, rather than simply reducing the total yield. Recognizing that unequal recovery may reflect substrate properties helps researchers interpret small-RNA measurements and sequencing results more cautiously.
Reaction conditions influence how effectively the ligase performs its activation and joining steps, although their effects depend on the RNA substrate. Conditions should therefore be evaluated together with end chemistry, sequence, and structure rather than treated as independent variables. Optimizing this combination can improve recovery and reduce systematic differences among RNA species.
Optimization focuses on improving joining while limiting preferential recovery of particular RNA molecules. Researchers can assess performance across substrates that differ in ends, sequence, or structure, then refine reaction conditions in light of those differences. The goal is not merely more ligated product, but a result that represents the starting RNA population more reliably.
During RNA sequencing library preparation, unequal joining can cause some RNA species to appear more frequently than others, while low-efficiency substrates may be underrepresented or missed. Evaluating and improving ligation performance helps reduce this distortion. Consequently, sequencing data can provide a more dependable view of transcript abundance, especially when RNA species occur at low levels.
Small-RNA analysis depends on capturing and joining short RNA molecules efficiently enough for accurate measurement. Differences in substrate ends, sequence, or structure can produce uneven recovery and obscure low-abundance species. Attention to ligation efficiency therefore supports more reliable comparisons among small RNAs and helps distinguish biological differences from technical losses.
For RNA circularization and synthetic transcript construction, successful joining determines whether the intended RNA product forms as expected. Inefficient or biased ligation can lower product recovery or favor particular substrates. Measuring performance under the relevant substrate and reaction conditions helps researchers judge product formation and improve the reliability of engineered RNA workflows.