Summary

Non Laser Capture Microscopy approccio per la Microdissezione delle Regioni discreti cervello di topo per la totale isolamento di RNA e valle Next-Generation Sequencing e profili di espressione genica

Published: November 13, 2011
doi:

Summary

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

Abstract

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.

Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-μm serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Protocol

1. Sampling setup and preparation Supplement Earle’s balanced salt solution (EBSS) with sodium bicarbonate (0.44 g per 100 mL EBSS) and glucose (0.884 g per 100 mL EBSS)9. Treat the EBSS with DEPC (0.1 mL per 100 mL EBSS) for at least 12 hours at 37 °C in an autoclavable bottle or flask with a screw top. Place 5 ¾” glass Pasteur pipets into a 1000 mL glass beaker, and place artist’s brushes, with bristles facing upward, into a 100 mL glass graduated cylinder. Add sufficient …

Discussion

There are many critical steps in this protocol that warrant careful attention and consideration. Selection of the appropriate tissue corer diameter must be determined for removing each respective region of interest. While the tissue corer diameters presented in Table 1 are suitable for collection of the listed brain regions, each investigator should determine the appropriate-sized tissue corer for optimal collection of their respective tissue(s) of interest. Secondly, the coronal section thickness reported in this met…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded by the Defense Advanced Research Projects Agency (DARPA) and the Army Research Office (ARO), award number W911NF-10-1-006.

Materials

Name of the reagent Company Catalogue number Comments
Isotemp 102 water bath Fisher Scientific 15-460-3Q  
RNase Zap Ambion AM9782  
Lab rotator (orbital shaker) Thermo Scientific 2314 – 1CEQ  
Pellet mixer VWR 47747-370  
RNase-free 0.5 mL pestle VWR KT749521-0590 Nuclease-free
Diethyl pyrocarbonate (DEPC) Sigma Life Science D5758-50ML  
Double-edge razor blades Stoelting 51427  
Stoelting tissue slicer with manual micrometer Stoelting 51425  
2/0 Round artist’s brush Princeton Art & Brush 4350R  
Harris Uni-Core tissue corer (0.75 mm) Ted Pella 15072  
Harris Uni-Core tissue corer (1.0 mm) Ted Pella 15099  
Harris Uni-Core tissue corer (1.5 mm) Ted Pella 15075  
Harris Uni-Core tissue corer (2.0 mm) Ted Pella 15076  
Guillotine Braintree Scientific RG-100  
DecapiCones Braintree Scientific MDC-200  
HyClone Earle’s balanced salt solution (EBSS) Thermo Scientific SH30029.02  
25 mL pipets Corning 4489 Nuclease-free
epT.I.P.S. LoRetention Dualfilter pipet tips, 20 – 300 μL Eppendorf 0030 077.636  
TrakMates screw top tubes with caps Thermo Scientific 3741  
VisionMate wireless 2D barcode reader Thermo Scientific 3122MTX  
VisionMate SR single rack 2D barcode reader Thermo Scientific 3115  
1-200 μL universal fit pipette tips Corning 4864 Nuclease-free
Cellstar cell culture dishes Greiner Bio-One 628160 Nuclease-free
Greiner Bio-One 96-well polystyrene Cellstar tissue culture plate, U-bottom USA Scientific 5665-0180 Nuclease-free
#4 Filter paper Whatman 1004 150  
Silicone bulb-type safety pipette filler Fisherbrand 13-681-102A  
NanoDrop 3300 Spectrophotometer Thermo Scientific ND-3300  

References

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Cite This Article
Atkins, N., Miller, C. M., Owens, J. R., Turek, F. W. Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling. J. Vis. Exp. (57), e3125, doi:10.3791/3125 (2011).

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