Gene expression profiling of neurochemically defined regions of the human brain by in situ hybridization-guided laser capture microdissection.
Laser capture microdissection (LCM) permits isolation of specific cell types and cell groups based upon morphology, anatomical landmarks and histochemical properties. This powerful technique can be used for region-specific dissection if the target structure is clearly delineated. However, it is difficult to visualize anatomical boundaries in an unstained specimen, while histological staining can complicate the microdissection process and compromise downstream processing and analysis. We now introduce a novel method in which in situ hybridization (ISH) signal is used to guide LCM on adjacent unstained sections to collect tissue from neurochemically defined regions of the human postmortem brain to minimize sample manipulation prior to analysis. This approach was validated in nuclei that provide monoaminergic inputs to the forebrain, and likely contribute to the pathophysiology of mood disorders. This method was used successfully to carry out gene expression profiling and quantitative real-time PCR (qPCR) confirmation from the dissected material. When compared to traditional micropunch dissections, our ISH-guided LCM method provided enhanced signal intensity for mRNAs of specific monoaminergic marker genes as measured by genome-wide gene expression microarrays. Enriched expression of specific monoaminergic genes (as determined by microarrays and qPCR) was detected within appropriate anatomical locations validating the accuracy of microdissection. Together these results support the conclusion that ISH-guided LCM permits acquisition of enriched nucleus-specific RNA that can be successfully used for downstream gene expression investigations. Future studies will utilize this approach for gene expression profiling of neurochemically defined regions of postmortem brains collected from mood disorder patients.
Bernard
René
R
Molecular & Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA. rene.bernard@charite.de
Kerman
Ilan A
IA
Meng
Fan
F
Evans
Simon J
SJ
Amrein
Irmgard
I
Jones
Edward G
EG
Bunney
William E
WE
Akil
Huda
H
Watson
Stanley J
SJ
Thompson
Robert C
RC
D000293:Adolescent; D000328:Adult; D000368:Aged; D000704:Analysis of Variance; D001921:Brain / Q000033:anatomy & histology / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000502:physiology; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D007834:Lasers; D008297:Male; D046228:Microarray Analysis / Q000379:methods; D042282:Microdissection / Q000379:methods*; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D011180:Postmortem Changes; D012333:RNA, Messenger / Q000378:metabolism
D009419:Nerve Tissue Proteins; D012333:RNA, Messenger