Collection of Frozen Rodent Brain Regions for Downstream Analyses

Jim Wager-Miller; Michelle Murphy Green; Hana Shafique; Ken Mackie

doi:10.3791/60474

Gromadzenie zamrożonych obszarów mózgu gryzoni do dalszych analiz

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Collection of Frozen Rodent Brain Regions for Downstream Analyses

Gromadzenie zamrożonych obszarów mózgu gryzoni do dalszych analiz

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07:06 min

April 23, 2020

DOI: 10.3791/60474-v

Jim Wager-Miller¹, Michelle Murphy Green¹, Hana Shafique¹, Ken Mackie¹

¹Department of Psychological and Brain Sciences, Gill Center for Biomolecular Research,Indiana University

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Overview

This procedure details the process of collecting discrete frozen brain regions to obtain high-quality protein and RNA. Using adult CD-1 wild type mice, the method allows for the preservation of target molecules during dissection, ensuring meticulous sample collection for downstream applications.

Key Study Components

Area of Science

Neuroscience
Biological Sample Processing
Protein and RNA Extraction

Background

Preserving brain tissues is crucial for molecular analysis.
Common brain landmarks assist in identifying regions of interest.
Frozen dissection methods enhance the integrity of samples.
Stored samples can undergo various analyses while retaining quality.

Purpose of Study

To establish a reliable method for obtaining high-quality RNA and protein from specific brain regions.
To validate the effectiveness of frozen dissection techniques.
To facilitate molecular investigations in neuroscience.

Methods Used

Utilized frozen brain regions from adult CD-1 wild type mice.
Maintained tissue at low temperatures during dissection to preserve integrity.
Key steps included flash freezing, thawing, and precise blade placements for clean cuts.
Used RIPA buffer for protein extraction and a guanidinium solvent for RNA extraction.

Main Results

The method yielded high-quality RNA with strong ribosomal banding after analysis.
Both frozen dissection and fresh harvesting methods provided RNA with high integrity numbers.
Protein integrity was validated through Western blotting, showing distinct bands.
This approach maintains sample morphology for accurate future comparisons.

Conclusions

The procedure enables high-quality molecular analysis of dissected brain regions.
It supports various downstream applications, contributing to neuroscience research.
This methodology aids in understanding how substances like THC affect brain development.

Frequently Asked Questions

What are the advantages of using frozen dissection for brain samples?

Frozen dissection preserves the integrity of tissues, ensuring high-quality molecular data. It allows researchers to maintain the microenvironment of the samples, which is crucial for accurate study.

How are regions of interest identified in the dissection process?

Regions of interest are identified using common brain landmarks from the Allen Mouse Brain Atlas. Careful alignment of the brain within the matrix aids in accurate dissection.

What types of data can be obtained from this method?

Researchers can obtain high-quality RNA and proteins suitable for various analyses, including RT-qPCR, RNA-Seq, and Western blot assays. These enable a deeper understanding of molecular changes in the brain.

How can this method be adapted for different applications?

The frozen dissection method can be tailored by adjusting the types of solvents used for extraction or by modifying the freezing and dissection protocols to fit specific research needs.

What considerations should be taken into account when using this dissection technique?

Maintaining low temperatures throughout the process is critical to preserving sample quality. Care must be taken to handle tissues gently to avoid degradation during dissection.

What is the expected timeline for this dissection method?

The initial freezing of the brain takes about 60 seconds, followed by a 10-minute equilibration period before dissection can begin. Subsequent steps depend on the complexity of operation and desired outcomes.

Ta procedura opisuje zbieranie dyskretnie zamrożonych obszarów mózgu w celu uzyskania wysokiej jakości białka i RNA przy użyciu tanich i powszechnie dostępnych narzędzi.

Procedura ta opisuje pobieranie dyskretnie zamrożonych obszarów mózgu w celu uzyskania wysokiej jakości białka i RNA przy użyciu niedrogich i powszechnie dostępnych narzędzi. Ponieważ obszary mózgu są utrzymywane w stanie zamrożonym od zbioru do sekcji, cząsteczki docelowe są zachowywane, a badacz ma czas na staranne przeanalizowanie i przechowywanie interesujących go regionów. Identyfikacja obszarów zainteresowania może początkowo być trudna.

Korzystanie ze wspólnych punktów orientacyjnych mózgu, gdy pojawiają się i cofają przez sekcje w odniesieniu do pożądanych regionów, sprawi, że identyfikacja będzie jasna. Po usunięciu mózgów od uśpionych dorosłych myszy typu dzikiego CD-1, należy błyskawicznie zamrozić tkankę na 60 sekund w ciekłym azocie lub izopentanie. Wstępnie schłodzić suchym lodem i przechowywać w temperaturze minus 80 stopni Celsjusza.

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