Obtaining high quality RNA from single cell populations in human postmortem brain tissue.
We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons. Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51 microg, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.
Pietersen
Charmaine Y
CY
Department of Structural and Molecular Neuroscience, McLean Hospital. cpietersen@mclean.harvard.edu
Lim
Maribel P
MP
Woo
Tsung-Ung W
TU
D001921:Brain / Q000166:cytology / Q000502:physiology*; D001923:Brain Chemistry; D020869:Gene Expression Profiling; D006801:Humans; D042282:Microdissection / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis; D017966:Pyramidal Cells / Q000737:chemistry*; D012313:RNA / Q000302:isolation & purification*
D012313:RNA
Nucleic Acids Res. 2005 Mar 30;33(6):e56
15800207