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Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting
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Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting.

Huifei Sophia Zheng1, Chen-Che Jeff Huang2

  • 1Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University; sophia.zheng@auburn.edu.

Journal of Visualized Experiments : Jove
|December 27, 2021
PubMed
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This study presents a Translating Ribosome Affinity Purification (TRAP) method to isolate cell-type-specific RNAs from EGFP-expressing cells in complex tissues. This technique enhances transcriptome analysis by overcoming cellular heterogeneity challenges.

Area of Science:

  • Molecular Biology
  • Genomics
  • Neuroscience

Background:

  • Cellular heterogeneity complicates transcriptome-level analysis of complex tissues.
  • Isolating cell-type-specific RNAs is crucial for accurate functional studies.
  • Existing methods may be limited by tissue complexity or cell sorting requirements.

Purpose of the Study:

  • To describe a protocol for isolating cell-type-specific RNAs from EGFP-expressing cells in complex tissues.
  • To enable transcriptome analysis without cell sorting using the Translating Ribosome Affinity Purification (TRAP) method.
  • To provide a method applicable to the NuTRAP mouse model and other EGFP-expressing systems.

Main Methods:

  • Translating Ribosome Affinity Purification (TRAP) using EGFP-expressing cells.

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  • Isolation of ribosome-bound RNAs from small cell populations within complex tissues.
  • Application of the protocol with the NuTRAP mouse model.

    • Main Results:

    • Successful isolation of cell-type-specific RNAs from EGFP-expressing cells.
    • Demonstration of the TRAP method's efficacy in complex tissues without cell sorting.
    • Validation of the protocol for use with the NuTRAP mouse model.

    Conclusions:

    • The described TRAP protocol effectively isolates cell-type-specific RNAs, addressing challenges of tissue heterogeneity.
    • This method facilitates robust transcriptome analysis from limited cell populations.
    • The protocol is versatile and applicable to various EGFP-expressing systems, including the NuTRAP mouse model.