Sin láser y de captura de enfoque de Microscopía de la microdisección de regiones discretas del cerebro del ratón para el aislamiento de ARN total y Downstream secuenciación de próxima generación y de perfiles de expresión génica

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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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Abstract

Materials

Name Company Catalog Number Comments
Isotemp 102 water bath Fisher Scientific 15-460-3Q
RNase Zap Ambion AM9782
Lab rotator (orbital shaker) Thermo Fisher Scientific, Inc. 2314 –- 1CEQ
Pellet mixer VWR international 47747-370
RNase-free 0.5 mL pestle VWR international KT749521-0590 Nuclease-free
Diethyl pyrocarbonate (DEPC) Sigma-Aldrich D5758-50ML
Double-edge razor blades Stoelting Co. 51427
St–lting tissue slicer with manual micrometer Stoelting Co. 51425
2/0 Round artist’s brush Princeton Art Brush 4350R
Harris Uni-Core tissue corer (0.75 mm) Ted Pella, Inc. 15072
Harris Uni-Core tissue corer (1.0 mm) Ted Pella, Inc. 15099
Harris Uni-Core tissue corer (1.5 mm) Ted Pella, Inc. 15075
Harris Uni-Core tissue corer (2.0 mm) Ted Pella, Inc. 15076
Guillotine Braintree Scientific, Inc. RG-100
DecapiCones Braintree Scientific, Inc. MDC-200
HyClone Earle’s balanced salt solution (EBSS) Thermo Fisher Scientific, Inc. 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 Fisher Scientific, Inc. 3741
VisionMate wireless 2D barcode reader Thermo Fisher Scientific, Inc. 3122MTX
VisionMate SR single rack 2D barcode reader Thermo Fisher Scientific, Inc. 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, Inc. 5665-0180 Nuclease-free
#4 Filter paper Whatman, GE Healthcare 1004 150
Silicone bulb-type safety pipette filler Fisher Scientific 13-681-102A
NanoDrop 3300 Spectrophotometer Thermo Fisher Scientific, Inc. ND-3300

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References

  1. Pickrell, J. K. Understanding mechanisms underlying human gene expression variation with RNA sequencing. Nature. 464, 768-772 (2010).
  2. Costa, V., Angelini, C., De Feis, I., Ciccodicola, A. Uncovering the complexity of transcriptomes with RNA-Seq. J. Biomed. Biotechnol. 853916, (2010).
  3. Nagalakshmi, U., Waern, K., Snyder, M. RNA-Seq: a method for comprehensive transcriptome analysis. Curr. Protoc. Mol. Biol. Chapter 4, 11-13 (2010).
  4. Anglicheau, D., Muthukumar, T., Suthanthiran, M. MicroRNAs: small RNAs with big effects. Transplantation. 90, 105-112 (2010).
  5. Forrest, A. R., Carninci, P. Whole genome transcriptome analysis. RNA. Biol. 6, 107-112 (2009).
  6. Twine, N. A., Janitz, K., Wilkins, M. R., Janitz, M. Whole transcriptome sequencing reveals gene expression and splicing differences in brain regions affected by Alzheimer's disease. PLoS One. 6, e16266-e16266 (2011).
  7. Paulsen, S. J. Gene expression profiling of individual hypothalamic nuclei from single animals using laser capture microdissection and microarrays. J. Neurosci. Methods. 177, 87-93 (2009).
  8. Winrow, C. J. Refined anatomical isolation of functional sleep circuits exhibits distinctive regional and circadian gene transcriptional profiles. Brain. Res. 1271, 1-17 (2009).
  9. Atkins, N. Circadian integration of glutamatergic signals by little SAAS in novel suprachiasmatic circuits. PLoS One. 5, e12612-e12612 (2010).
  10. Technologies, L. MagMAX-96 Total RNA Isolation Kit Instruction Manual. Forthcoming.

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