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Related Concept Videos

RNA-seq03:21

RNA-seq

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. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
Ribosome Profiling02:24

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...
Alternative RNA Splicing02:18

Alternative RNA Splicing

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...
Alternative RNA Splicing02:18

Alternative RNA Splicing

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...
RNA Splicing01:32

RNA Splicing

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...
RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...

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Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models
09:58

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Published on: December 9, 2016

Alternative expression analysis by RNA sequencing.

Malachi Griffith1, Obi L Griffith, Jill Mwenifumbo

  • 1Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada.

Nature Methods
|September 14, 2010
PubMed
Summary
This summary is machine-generated.

We developed ALEXA-seq, a novel RNA sequencing method to analyze mRNA isoforms. This method revealed global splicing disruption in fluorouracil-resistant colorectal cancer cells, identifying new mRNA variants.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Alternative splicing generates diverse mRNA isoforms from a single gene.
  • Understanding alternative splicing is crucial for cell function and disease.
  • Cancer cells often exhibit altered gene expression and splicing patterns.

Purpose of the Study:

  • To develop and validate a novel RNA sequencing method, ALEXA-seq, for cataloging and analyzing mRNA isoforms.
  • To investigate differential and alternative mRNA isoform expression in cancer.
  • To compare gene expression in fluorouracil-resistant versus nonresistant colorectal cancer cell lines.

Main Methods:

  • Development of ALEXA-seq for massively parallel RNA sequencing data analysis.
  • Application of ALEXA-seq to compare human colorectal cancer cell lines with differing fluorouracil resistance.
  • Validation of ALEXA-seq results using exon tiling microarrays, splicing microarrays, RT-PCR, qPCR, and Sanger sequencing.

Main Results:

  • ALEXA-seq successfully cataloged transcripts and assessed differential and alternative expression of mRNA isoforms.
  • Global disruption of splicing was observed in fluorouracil-resistant colorectal cancer cells.
  • New mRNA isoforms resulting from exon skipping, alternative splice site usage, and intron retention were identified in resistant cells.

Conclusions:

  • ALEXA-seq is a sensitive and specific method for analyzing mRNA isoforms.
  • Fluorouracil resistance in colorectal cancer is associated with widespread alterations in alternative splicing.
  • The findings provide insights into the molecular mechanisms underlying drug resistance in cancer.