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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.
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
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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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Methods for Characterization of Alternative RNA Splicing.

Samuel E Harvey1, Chonghui Cheng2

  • 1Division of Hematology/Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA.

Methods in Molecular Biology (Clifton, N.J.)
|January 2, 2016
PubMed
Summary

Detecting gene splice variants requires precise PCR primer design for accurate quantification of alternative splicing. Splicing minigenes in cell culture aid in studying alternative splicing regulation.

Keywords:
Alternative splicingMinigeneRNART-PCRSplicing factorsSplicing regulationVariable exon

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Alternative splicing is a key mechanism for increasing proteomic diversity.
  • Understanding alternative splicing is crucial for comprehending gene expression regulation and its role in disease.

Purpose of the Study:

  • To describe methods for quantifying alternative splicing events.
  • To introduce a splicing minigene system for studying alternative splicing regulation.

Main Methods:

  • Quantitative real-time PCR (RT-qPCR) and semi-quantitative PCR for detecting splice isoform abundance in total RNA.
  • Development and utilization of a splicing minigene system in mammalian cell culture.

Main Results:

  • RT-PCR methods enable the quantification of differentially spliced isoforms.
  • Careful primer design is essential for specific detection of splice variants.
  • The splicing minigene system facilitates the investigation of regulatory mechanisms controlling alternative splicing.

Conclusions:

  • RT-PCR provides a robust approach for quantifying alternative splicing.
  • Splicing minigenes are valuable tools for dissecting the regulation of alternative splicing at specific exons.