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Related Concept Videos

RNA-seq03:21

RNA-seq

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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. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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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.
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Rup (RNA-seq Usability Assessment Pipeline) - Quality Control for Bulk RNA-seq Experiments in Eukaryotes
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Proteomic Validation of Transcript Isoforms, Including Those Assembled from RNA-Seq Data.

Aidan P Tay1,2, Chi Nam Ignatius Pang1,2, Natalie A Twine1,2

  • 1Systems Biology Initiative, The University of New South Wales , Sydney, New South Wales 2052, Australia.

Journal of Proteome Research
|May 12, 2015
PubMed
Summary
This summary is machine-generated.

Identifying protein isoforms is crucial for human proteome analysis. This study integrates RNA-seq and mass spectrometry data to detect protein isoforms, finding many detectable ones, though some remain challenging.

Keywords:
RNA-seqalternative splicingisoform-specific peptidesmesenchymal stem cellproteotypic peptidessplice-junction peptides

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Area of Science:

  • Proteomics
  • Transcriptomics
  • Genomics

Background:

  • Human proteome analysis necessitates understanding protein isoforms.
  • Previous work introduced the PG Nexus pipeline for exon and splice junction validation using integrated genomics and proteomics data.

Purpose of the Study:

  • To comprehensively explore the identification of protein isoforms using RNA-sequencing (RNA-seq) transcriptomics and proteomic analysis from the same human samples.
  • To evaluate the effectiveness of the TranscriptCoder tool in generating a protein isoform sequence database for mass spectrometry (MS/MS) data matching.

Main Methods:

  • Analysis of RNA-seq data from human mesenchymal stem cells (hMSC) using the TranscriptCoder tool to create a protein isoform database.
  • Matching MS/MS data from hMSC samples against the TranscriptCoder-derived database, Ensembl, and neXtProt.
  • Covisualization of messenger RNA (mRNA) isoforms and peptides in a genome browser.

Main Results:

  • Unambiguous identification of approximately 450 protein isoforms with isoform-specific proteotypic peptides using TranscriptCoder or Ensembl databases.
  • Identification of candidate hMSC-specific isoforms for DPYSL2 and FXR1 genes.
  • Demonstration that groups of non-isoform-specific peptides can identify many isoforms, and that targeted MS/MS assays can detect them.
  • Revelation that some isoforms are difficult to identify unambiguously due to a lack of distinguishing peptides.

Conclusions:

  • Integration of RNA-seq and proteomics effectively identifies numerous protein isoforms.
  • The TranscriptCoder tool aids in the discovery and validation of protein isoforms.
  • Challenges remain in the unambiguous identification of certain protein isoforms, highlighting areas for future research.