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Related Concept Videos

MicroRNAs01:22

MicroRNAs

MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends...
MicroRNAs01:22

MicroRNAs

MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...
MicroRNAs01:22

MicroRNAs

MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends...
RNA Interference01:23

RNA Interference

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
Experimental RNAi02:15

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
siRNA - Small Interfering RNAs02:30

siRNA - Small Interfering RNAs

Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...

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Related Experiment Video

Updated: May 23, 2026

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools
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A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

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A simplified method for constructing artificial microRNAs based on the osa-MIR528 precursor.

Fei Yan1, Yuwen Lu, Gentu Wu

  • 1State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.

Journal of Biotechnology
|April 3, 2012
PubMed
Summary

Researchers simplified artificial microRNA (amiRNA) construction using a single PCR step, making gene silencing in plants more efficient. This new method targets the UPF1 gene in Nicotiana benthamiana, demonstrating effective expression silencing.

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MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method

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Area of Science:

  • Plant molecular biology
  • RNA interference
  • Gene silencing technologies

Background:

  • Artificial microRNAs (amiRNAs) are effective tools for gene silencing in plants.
  • Current methods, like Schwab et al., require multiple PCR steps, increasing complexity.
  • A simplified and efficient amiRNA construction protocol is needed.

Purpose of the Study:

  • To develop a simplified one-step PCR method for constructing artificial microRNAs (amiRNAs).
  • To optimize the use of the osa-MIR528 backbone for amiRNA synthesis.
  • To validate the functionality of amiRNAs constructed using this method.

Main Methods:

  • A single-step PCR strategy was employed by adding prolonging sequences to primers.
  • The osa-MIR528 precursor was utilized as a backbone for amiRNA construction.
  • The optimal length of the prolonging sequence for the osa-MIR528 precursor was determined.
  • An amiRNA targeting the Nicotiana benthamiana UPF1 gene was constructed and tested.

Main Results:

  • A simplified one-step PCR method for amiRNA construction was successfully developed.
  • The required length of the prolonging sequence in the osa-MIR528 precursor was identified.
  • The constructed amiRNA effectively silenced the expression of the UPF1 gene in Nicotiana benthamiana leaves.
  • Transient expression confirmed the functionality of the amiRNA in gene silencing.

Conclusions:

  • The novel one-step PCR method offers a significant simplification for amiRNA construction.
  • This streamlined approach enhances the efficiency of developing gene silencing tools for plant research.
  • The validated amiRNA targeting UPF1 demonstrates the method's potential for functional genomics studies in plants.