Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression.
Laser capture microdissection (LCM) is increasingly being used in quantitative gene expression studies of the nervous system. The current study aimed at determining the impact of various tissue manipulations on the integrity of extracted RNA in LCM studies. Our data indicate that various tissue preparation strategies prior to microdissection may decrease RNA quality by as much as 25%, thus affecting expression profiles of some genes. To circumvent this problem, we developed a strategy for reverse transcriptase real-time PCR that has considerable sensitivity and can be used to calculate relative changes in gene expression. This approach was validated in subregions of the rat cerebellum. Accordingly, expression of glial gene markers - myelin-associated glycoprotein and proteolipid protein 1 - was found 70-160-fold higher in the white matter layer of the cerebellar cortex as compared to the neuron-enriched granular layer. In contrast, expression of a specific neuronal maker, neuron-specific enolase, was found seven-fold higher in the granular layer, as compared to the white matter layer. Furthermore, this approach had high sensitivity and specificity as we were able to detect a 38% decrease in the expression of neuron-specific enolase without a change in the expression of glial markers following administration of the neurotoxin, ibotenic acid. These results demonstrate feasibility of performing accurate semi-quantitative gene expression analyses in LCM samples.
Kerman
Ilan A
IA
Molecular and Behavioral Neuroscience Institute, Department of Psychiatry, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109, USA. kerman@umich.edu
Buck
Bradley J
BJ
Evans
Simon J
SJ
Akil
Huda
H
Watson
Stanley J
SJ
D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D053685:Laser Therapy / Q000379:methods*; D042282:Microdissection / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011786:Quality Control; D012313:RNA / Q000032:analysis* / Q000235:genetics*; D051381:Rats; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D046509:Tissue Culture Techniques / Q000379:methods*
D009419:Nerve Tissue Proteins; D012313:RNA