Quantification of layer-specific gene expression in the hippocampus: effective use of laser microdissection in combination with quantitative RT-PCR.
Laser microdissection in combination with quantitative RT-PCR is now widely appreciated as an excellent tool for quantifying mRNA levels in defined cell populations. It may be particularly useful in the hippocampal formation, where principal cells form distinct and readily identifiable cell layers. Here we are presenting an optimized protocol for labeling hippocampal principal cells on foil-mounted sections for microdissection with the Leica AS LMD system and discuss potential further applications and pitfalls. Employing this optimized method, we studied changes in brain-derived neurotrophic factor (BDNF) mRNA expression in granule cells of the mouse dentate gyrus following unilateral entorhinal cortex lesion. In this lesioning paradigm, changes in BDNF mRNA expression have previously been reported in the rat. Using laser microdissection, the granule cell layers ipsi- and contralateral to the lesion were collected and changes in BDNF levels were quantified using quantitative RT-PCR. BDNF mRNA levels were five-fold higher on the ipsilateral side compared to levels found on the contralateral side or in controls. The development of this optimized method for laser microdissection and subsequent quantitative RT-PCR allows layer-specific quantification of gene expression levels in the hippocampus and may be similarly employed in other brain areas or tissues with a laminar arrangement or high density of cells.
Burbach
Guido J
GJ
Institute of Clinical Neuroanatomy, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Burbach@em.uni-frankfurt.de
Dehn
Doris
D
Del Turco
Domenico
D
Deller
Thomas
T
D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006651:Histocytochemistry / Q000379:methods; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D019208:Brain-Derived Neurotrophic Factor; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S