トータルRNAの単離およびダウンストリーム次世代シーケンシングと遺伝子発現プロファイリングのための離散マウスの脳領域のマイクロダイセクションのための非レーザーキャプチャー顕微鏡のアプローチ

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 RNAs^2-4. 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 excision^7,8. 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…

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