Home › Biochemistry › In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation
Biochemistry JoVE (Open Access) Citable · DOI

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

DOI: 10.3791/51255-v
What you'll learn
  • ✓ Perform polyribosome fractionation on CNS tissue samples
  • ✓ Isolate ribosome-bound RNA versus total RNA fractions
  • ✓ Assess translational and transcriptional regulation in vivo
  • ✓ Analyze translatome differences between brain and spinal cord
Protocol

Biopharma Insights This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Difficulty
advanced
Total time
~4–6 hours per sample (tissue preparation through RNA isolation)
Model organism
Mouse
Biosafety
BSL-1

Steps

1
Prepare spinal cord and brain tissue

Extract and rapidly freeze CNS tissue from euthanized mice to preserve translatome composition. Tissue must be processed quickly to minimize RNA degradation and translational changes.

▶ 01:45
2
Perform myelin flotation

Remove myelin and other lipid-rich contaminants from CNS tissue homogenates using flotation separation. This step purifies the sample for downstream polyribosome fractionation.

▶ 03:28
3
Fractionate polyribosomes on sucrose gradient

Separate ribosomal complexes (monosomes, light polysomes, heavy polysomes) by ultracentrifugation through a sucrose density gradient. Each fraction represents different translational activity states.

▶ 04:33
4
Isolate RNA from individual fractions

Extract total and ribosome-bound RNA from each polyribosome fraction. Quantify RNA yields to assess translation burden across different polysome populations.

▶ 06:23
5
Analyze RNA profiles from tissue types

Compare polyribosome fractionation patterns and RNA distributions between brain and spinal cord. Differences reveal tissue-specific translational regulation in vivo.

▶ 07:36
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