Journal of Neuroscience Methods 177 (2009) 87–93 Contents lists available at ScienceDirect Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth Gene expression profiling of individual hypothalamic nuclei from single animals using laser capture microdissection and microarrays Sarah Juel Paulsen a,b,∗ , Leif Kongskov Larsen a , Jacob Jelsing a , Uwe Janßen c , Bernhard Gerstmayer c , Niels Vrang a a Rheoscience A/S, Glerupvej 1, DK-2610 Rødovre, Denmark b University of Southern Denmark, BMB, Campusvej 55, DK-5230 Odense M, Denmark c Miltenyi Biotec GmbH, Friedrich-Ebert-Straße 68, 51429 Bergisch Gladbach, Germany article info Article history: Received 26 June 2008 Received in revised form 18 September 2008 Accepted 26 September 2008 Keywords: Laser capture microdissection Microarray analysis Hypothalamic arcuate nucleus Food deprivation abstract In order to identify novel genes involved in appetite and body weight regulation we have developed a microarray based method suitable for detecting small changes in gene expression in discrete groups of hypothalamic neurons. The method is based on a combination of stereological sampling, laser capture microdissection (LCM), PCR based amplification (SuperAmp TM ), and one-color cDNA microarray analysis. To validate the method we assessed and compared fasting induced changes in mRNA levels of Neuropep- tide Y (NPY) and proopiomelanocortin (POMC) in the hypothalamic arcuate nucleus (ARC) of diet-induced obese rats using cDNA microarrays, quantitative PCR and in situ hybridization. All methods revealed statis- tically significant fasting-induced changes in NPY and POMC expression. An additional 3480 differentially expressed probes (fold change >1.22, t-test p = 0.05) were identified in the microarray analysis. Our findings demonstrate a consistent gene expression pattern across three different gene expres- sion detection methods and strongly suggest that LCM coupled microarray analysis combined with SuperAmp TM can be used as a semi-quantitative mRNA profiling tool. Importantly, the sensitivity of the method greatly improves the usefulness of the microarray technology for gene expression profiling in non-homogeneous tissues such as the brain. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Although the microarray technology has surpassed its 10 years anniversary, the full potential of the technology as a target discov- ery engine remains to be unravelled. The use of cDNA microarrays has expanded greatly as the quality of data has increased, and particularly commercial one-dye platforms generate very consis- tent results (Kuo et al., 2006). Laser capture microdissection has been introduced as an efficient and precise method to sample sub- groups of cells in heterogeneous tissues such as the brain. Even though it seems natural to combine array technologies with the LCM techniques, the minute amounts of tissue – and hence useful RNA – obtained during LCM has hampered such an approach (at least on individual test samples). Recently, however, a PCR based amplification method (SuperAmp TM ) was demonstrated to gen- erate sufficient quantities of cRNA/cDNA for microarray analysis ∗ Corresponding author at: Rheoscience A/S, Glerupvej 1, DK-2610 Rødovre, Den- mark. Tel.: +45 44501975; fax: +45 44501962. E-mail address: sjp@rheoscience.com (S.J. Paulsen). from as little as 100–1000 cells (Appay et al., 2007). With these technological advances in mind the present study was initiated to develop a method for microarray profiling of discrete hypothala- mic nuclei from individual rats. Especially, the ability to perform array analysis without prior pooling of tissue would avoid data interpretation limitations from pooled tissue samples, i.e. allow the possibility to exclude biological outliers and hence enable corre- lations between phenotype and gene expression. In addition, we wanted to develop a method sensitive enough for detecting rela- tively small gene expression changes. 2. Materials and methods 2.1. Animals Sixteen male diet-induced obese rats (app. 22 weeks old; Rheoscience in-house breeding colony) were maintained under a 12/12 LD cycle (lights on at 06:00) with stable temperature (22 ± 1 ◦ C) and humidity (50 ± 5%) conditions. Animals were housed individually. During acclimatization the rats had ad libitum access to an energy-dense high-fat diet (HE; 4.41 kcal/g—energy 0165-0270/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2008.09.024