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Related Concept Videos

Alternative RNA Splicing02:18

Alternative RNA Splicing

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Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
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Related Experiment Video

Updated: Jun 24, 2025

Author Spotlight: Polysome Profiling Protocol for Studying Translational Regulation in Arabidopsis Under Heat Stress
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A Method to Quantitatively Examine Heat Stress-Induced Alternative Splicing in Plants by RNA-Seq and RT-PCR.

Yu Ling1, Yujian Mo1, Shanlan Chen1

  • 1College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang, People's Republic of China.

Methods in Molecular Biology (Clifton, N.J.)
|June 13, 2024
PubMed
Summary

This study introduces a new method combining RNA-sequencing and RT-PCR to detect heat stress-induced alternative splicing (AS) events in plants. This approach effectively visualizes and validates AS, particularly intron retention, in species like Arabidopsis thaliana.

Keywords:
Alternative splicingBAM fileIGV programRNA sequencingRT-PCRSnapgeneStress

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Genetics

Background:

  • Alternative splicing (AS) is a key post-transcriptional regulation in eukaryotes, increasing mRNA diversity.
  • Intron retention is the predominant AS form in plants, often upregulated under environmental stress.
  • Current methods for AS detection in plants are often complex or inadequate.

Purpose of the Study:

  • To develop and present a robust method for visualizing and validating heat stress-induced AS events in plants.
  • To address the limitations of existing techniques for studying plant AS.
  • To demonstrate the method's efficacy using Arabidopsis thaliana and the HEAT SHOCK PROTEIN21 (HSP21) gene.

Main Methods:

  • Integration of RNA-sequencing (RNA-seq) data with reverse transcription PCR (RT-PCR).
  • Application to study heat stress responses in Arabidopsis thaliana.
  • Focus on validating alternative splicing events, specifically intron retention.

Main Results:

  • Successful visualization and validation of heat stress-induced AS events.
  • Demonstrated the utility of the combined RNA-seq and RT-PCR approach in plant systems.
  • Identified AS events in the HSP21 gene of Arabidopsis thaliana under heat stress.

Conclusions:

  • The developed method offers a practical solution for studying AS in plants, overcoming previous technical challenges.
  • This technique enhances the understanding of plant responses to environmental stress at the post-transcriptional level.
  • Provides a reliable platform for future research into plant AS mechanisms.