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Related Concept Videos

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...

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Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
10:00

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Published on: October 28, 2014

Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments.

M Julieta del Prete1, Rolando Vernal, Helmut Dolznig

  • 1Department of Immunology, Centro de Investigaciones Biológicas, Madrid, Spain.

RNA (New York, N.Y.)
|January 24, 2007
PubMed
Summary

Researchers developed a new method to isolate polysome-bound mRNA from small solid tissue samples. This technique overcomes challenges with heparin inhibition, enabling gene expression analysis in vivo.

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Area of Science:

  • Molecular Biology
  • Gene Expression Regulation
  • Translational Control

Background:

  • Translation control is crucial for gene expression in cells.
  • Its role in vivo (tissues/organs) is unclear due to limited methods for solid tissue RNA analysis.
  • Existing methods struggle with small sample sizes and RNA integrity.

Purpose of the Study:

  • To develop an efficient method for polysome-bound messenger RNA (mRNA) fractionation from small solid tissue samples.
  • To enable the study of translational control in vivo.
  • To optimize RNA isolation for downstream applications like RT-PCR.

Main Methods:

  • Solid tissue samples were pulverized in liquid nitrogen and lysed.
  • Cytoplasmic extracts were fractionated using sucrose gradients.
  • Heparin, an inhibitor, was removed using lithium chloride precipitation.
  • RNA was prepared from fractions for downstream analysis.

Main Results:

  • A reliable method was established for preparing high-quality polysome-bound mRNA from small tissue samples (10-20 mg).
  • Heparin was identified as an inhibitor of reverse transcription and successfully removed.
  • The method is effective for both human and mouse tissues.
  • Prepared RNA is suitable for Northern blotting, RT-PCR, and expression profiling.

Conclusions:

  • A novel, efficient method allows polysome-bound mRNA isolation from small solid tissue samples.
  • This technique facilitates the study of in vivo translational gene regulation.
  • The optimized protocol supports various downstream molecular analyses.