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This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.
As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5–1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 °C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.
This work illustrates the dissection of frozen brain regions for extraction of high-quality nucleic acid or protein using a reference, such as the Allen Mouse Brain Atlas1, as a guide. In this technique, brains are flash-frozen and stored at -80 °C for later sectioning and dissection while being maintained in a frozen condition. This process allows the researcher to harvest a large number of brains in one session and later dissect them for an accurate collection of multiple brain regions.
The accurate collection of brain regions of interest (ROIs) is often required when answering questions related to gene and pr....
Animals used in this study were treated in an ethical and humane manner as set forth by Indiana University’s Institutional Animal Care and Use Committee (IACUC) and National Institutes of Health (NIH) guidelines.
NOTE: All tools and surfaces should be washed with an appropriate solvent to remove nucleases18 before starting any work.
1. Storing brains
In order to validate this method, the medial prefrontal cortex was collected from adult CD1 wildtype male mice and RNA and protein were extracted and characterized. RNA was analyzed by capillary electrophoresis. Degraded RNA displays a loss in the intensity of the 28S and 18S ribosomal bands and also shows degradation products as a smear between 25 and 200 nucleotides (Figure 5A, sample 1). High quality RNA shows distinct ribosomal bands with little to no signal in the lower molecular weight.......
This work describes a technique to isolate small, specific regions of brain while limiting degradation of nucleic acid and protein. Damage to brain tissues happens quickly once an organism dies. This is partially due to a rapid buildup of extracellular glutamate and the resultant excitotoxicity that occurs21. Messenger RNA is particularly vulnerable to degradation22,23. Breakdown of protein and nucleic acid is greatly reduced at low temper.......
This work was supported by the NIH, DA043982 and DA046196.
....Name | Company | Catalog Number | Comments |
0.5 mm Mouse coronal brain matrice | Braintree Scientific | BS-SS 505C | Cutting block |
0.5 mm Rat coronal brain matrice | Braintree Scientific | BS-SS 705C | Cutting block |
1.0 mm Biopsy Punch with plunger | Electron Microscopy Sciences | 69031-01 | |
1.5 mL microcentrifuge tubes | Dot Scientific | 229443 | For storing frozen ROIs |
1.5 mm Biopsy Punch with plunger | Electron Microscopy Sciences | 69031-02 | |
2.0 mm Biopsy Punch with plunger | Electron Microscopy Sciences | 69031-03 | |
4-12% NuPage gel | Invitrogen | NPO323BOX | protein gradient gel |
Bioanalyzer System | Agilent | 2100 | RNA analysis system |
Dounce tissue grinder | Millipore Sigma | D8938 | Glass tissue homogenizer |
Dry Ice | |||
Fiber-Lite | Dolan-Jenner Industries Inc. | Model 180 | Cool lamp |
Glass plates | LabRepCo | 11074010 | |
HALT | ThermoFisher | 78440 | protease inhibitor cocktail |
Low profile blades | Sakura Finetek USA Inc. | 4689 | |
mouse anti-actin antibody | Developmental Studies Hybridoma Bank | JLA20 | Antibody |
Nanodrop | Thermo Scientific | 2000C | Used in initial RNA purity analysis |
No. 15 surgical blade | Surgical Design Inc | 17467673 | |
Odyssey Blocking buffer | LiCor Biosciences | 927-40000 | Western blocking reagent |
Omni Tissue Master 125 | VWR | 10046-866 | Tissue homogenizer |
rabbit anti-KCC2 antibody | Cell Signaling Technology | 94725S | Antibody |
RNA Plus Micro Kit | Qiagen | 73034 | Used to extract RNA from small tissue samples |
RNaseZap | Life Technologies | AM9780 | |
Scalpel handle | Excelta Corp. | 16050103 | |
Standard razor blades | American Line | 66-0362 | |
TRIzol Reagent | ThermoFisher Scientific | 15596026 | Used to extract RNA from tissue |
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