Gene profiling of laser-microdissected brain regions and sub-regions

Brain Research Protocols 15 (2005) 66 – 74 www.elsevier.com/locate/brainresprot

Protocol

Gene profiling of laser-microdissected brain regions and sub-regions Pietro Paolo Sanna*, Alvin R. King, Lena D. van der Stap, Vez Repunte-Canonigo Department of Neuropharmacology, The Scripps Research Institute, 10550 N. Torrey Pines Road, CVN-12 La Jolla, CA 92037, USA Accepted 7 April 2005 Available online 8 June 2005

Abstract The application of transcriptomics and proteomics approaches to accurately dissected anatomically-defined brain regions and sub-regions remains a central focus of current neurobiological investigations as well as a necessary step towards single-neuron neurogenomics and neuroproteomics. A protocol is described for the simple, rapid, and reproducible laser microdissection of brain regions and sub-regions for microarray-based gene expression analyses from individual rats or mice using two rounds of in vitro transcription (IVT). The results presented also demonstrate that the current Affymetrix GeneChip\ arrays are well suited for this experimental design with high reproducibility and limited effects of the shortening of target RNA caused by the double IVT approach. D 2005 Elsevier B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Gene structure and function: general Keywords: Laser microdissection; Laser capture microdissection; Laser pressure catapulting; Microarray; Array; Gene profiling; Expression; mRNA; Proteomics; RAE230A; Rat 230A; Rat 230B; Rat Genome 230 2.0; Rat 230 2.0; RNU34; Rat Neurobiology U34; Mouse Genome 430 2.0; Mouse Expression Set 430; Murine 11K Set; MG_U74; MOE430; Mu11k

1. Type of research The study of neuroscience and neuropharmacology has greatly benefited from advances in techniques for the microdissection of anatomically-defined brain regions and sub-regions. The introduction of technologies for the largescale analysis of mRNA and protein content has now made it possible to extend these studies to genome-wide transcriptomics and proteomics analyses of physiological and pathological conditions. Ultimately, it would be highly desirable to study the transcriptome and proteome of individual cells or defined cell populations [11,16,19]. However, the depth of our knowledge is, in many cases, still insufficient to factor in the cellular heterogeneity of neuronal populations. Therefore, there is a need for simple reproducible protocols to analyze gene expression changes in anatomically-defined brain regions and sub-regions. The * Corresponding author. E-mail address: [email protected] (P.P. Sanna). 1385-299X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainresprot.2005.04.002

present protocol was designed for speed and ease of execution to allow one to monitor gene expression changes in multiple regions in large behavioral experiments comprising various conditions with an adequate statistical sample size (n). Several techniques exist for the dissection of discrete brain regions and sub-regions. These include, among others, freehanded dissections, manual punches, and scalpel dissections from macro-slices and cryostat or vibratome slices, as well as the use of modified microscopes – introduced over the last few years – outfitted with lasers to dissect and collect small specimens from tissue sections. These various dissection strategies differ in their anatomical accuracy, execution time, and they yield considerably different amounts of RNA or proteins. Some of the manual dissection methods still retain great utility for certain applications. While entirely manual dissections usually result in excessive variability, some of the aided manual dissections allow for acceptable accuracy and yield good amounts of proteins or RNA. For

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instance, manual punching from 1- to 2-mm-thick macroslices obtained with a wire brain slicer or a mold fitted with slots for razor blades is an effective technique when a large number of regions need to be dissected in a short period of time with reasonably good reproducibility (Fig. 1A). The resolution limit of this technique is usually about 0.5– 1 mm, depending on the region and experience of the operator. This dissecting technique yields RNA or protein samples large enough to conduct microarray analyses with a single round of in vitro transcription (IVT), which requires 1– 15 Ag of RNA [3], or conventional analytical 2D gels that typically require 50 – 100 Ag or more [23]. Despite the widespread use of this dissecting technique, most of the brain regions and subregions involved in learning and plasticity, pathogenesis of disease, or the actions of drugs of abuse cannot be manually dissected in the rat brain without including some of the surrounding tissue. A greater anatomical resolution can be achieved with a combination of scalpel and punching dissections of cryostat sections [20], but this is a considerably more time-consuming strategy and it is sometimes complicated by the difficulty of reliably identifying the boundaries of brain regions and sub-

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regions on frozen sections. A further improvement on manual dissection strategies is the dissection of transilluminated macro-slices of 100 – 300 Am thickness obtained with a vibratome or tissue chopper, as described by Cuello and Carson [10]. Although labor intensive, this approach has greater anatomical resolution than other manual dissection techniques (Figs. 1B, C). Recent studies highlight how biologically meaningful RNA and protein changes in the brain are often of limited magnitude (see, for instance, [13,17,24,25]). Therefore, greater anatomical accuracy is required to improve the ability of genomic and proteomic approaches to detect meaningful differences. Higher anatomical accuracy in the dissections can ultimately be achieved by laser microdissection. The main characteristics and features of some of the currently available laser-dissection systems are summarized in Table 1. The makers’ websites are listed to facilitate further research into their characteristics. All of these systems are highly effective at what they were designed to do, and users may select the system appropriate for their specific research application. For the present application (microdissection of rodent brain regions and sub-regions), we have used the Leica AS LMD (Figs. 1D, 2, and 3). The

Fig. 1. Brain microdissection techniques. (A) Manual punching of a brain macro-slice. The area of the rat locus ceruleus was punched from a 1-mm brain slice using a modified needle outfitted with a plunger to facilitate the transfer of the dissected tissue. Manual punching from 1- to 2-mm-thick macro-slices obtained with a wire brain slicer or a mold fitted with slots for razor blades remains a useful dissection technique. This technique is especially useful when a large number of regions need to be dissected in a short period of time at the sacrifice of anatomical resolution. The resolution limit of this technique is usually about 0.5 – 1 mm, depending on the region and experience of the operator. (B) Manual microdissection of transilluminated brain slices. A coronal brain slice (300 Am) through the bed nucleus of the stria terminalis (BNST) was obtained with a vibratome (Campdem Instruments MA752), and it is shown through the lens of a stereo dissection microscope (Zeiss Stemi SV6) outfitted with transillumination according to the technique of Cuello and Carson [10]. (C) The dorsolateral BNST (*) was manually microdissected from the transilluminated brain slices shown in panel A. Microdissections of transilluminated macro-slices of 100 – 300 Am thickness obtained with a vibratome or tissue chopper [10] have greater anatomical resolution than other manual dissection techniques. (D) Laser microdissection of brain slices (25 Am) for microarray gene profiling. Dissected regions were: A, lateral and basolateral nuclei of the amygdala; B, central nucleus of the amygdala; C, medial nucleus of the amygdala; D, lateral hypothalamic area; E, anterior hypothalamic area; F, paraventricular nuclei of the hypothalamus; G, retrochiasmatic area and periventricular nucleus of the hypothalamus. Scale bars = 2 cm.

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Table 1 Laser dissection and manipulation devices Manufacturer

Device(s) \

Specimen type

Primary applications

Website

Arcturus

PixCell IIe LCM AutoPixi Automated LCM

Fixed or frozen Fixed or frozen

Microdissection Microdissection (automated a)

http://www.arctur.com/

Leica

AS LMD

Fixed or frozen

Microdissection (computer controlled, automated a)

http://www.light-microscopy.com/

Microdissection (computer controlled)

http://www.palm-mikrolaser.com/

Live cell cutting module P.A.L.M.

MMI

PALM\ EasyBeam

Fixed or frozen

PALM\ MicroBeam PALM\ MicroBeam HT CombySystem Module

Fixed or frozen Fixed or frozen Live cells

Cellcut\

Fixed or frozen or Live cells Live cells

Cellector\ Module Cellmanipulator\ Module Cell Robotics International

LaserScisscors\ LaserTweezers\

a

Live cells

Live cells Fixed or frozen or Live cells Live cells

Microdissection (automated a) Transfer/manipulation of single cells/organelles Microdissection (computer controlled) Transfer/manipulation of single cells Micromanipulation of cells

http://www.molecular-machines.com

Microdissection

http://www.cellrobotics.com

Transfer/manipulation of single cells/organelles

Cell recognition capability.

dissection of brain sub-regions can also be carried out with other devices listed in Table 1.

2. Materials RNAlater (Ambion) 2-Methylbutane Ethanol/dry ice bath PENfoil slides (Leica) Optional: Graded ethanol Arcturus Picopure RNA Isolation Kit (Arcturus) RiboAmp OA RNA Amplification Kit (Arcturus) BioArray High Yield RNA Transcript Labeling Kit (Enzo) GeneChip\ high-density oligonucleotide arrays (Affymetrix)

3. Detailed procedure 3.1. Tissue preparation Tissue dehydration alone provides some protection from degradation of RNA. However, the quality of the recovered RNA can vary. Therefore, we have experimented with various strategies to preserve RNA for laser microdissection of brain tissue. We have settled on perfusing the animals intracardiacly with a diluted solution

of RNAlater (Ambion), a commercial RNA protectant, in ice-cold PBS. While for optimal protection, this product needs to be used undiluted. The density of the undiluted solution makes it ineffective for perfusion, because it fails to penetrate the brain microvasculature. Additionally, since it causes considerable shrinkage of the tissue, immersion of the brains in undiluted RNAlater greatly complicates the subsequent dissection due to the difficulty of reliably and reproducibly identifying the brain regions of interest in the shrunken tissue. We found that perfusion with a 10% dilution of RNAlater in PBS reduces RNA degradation. Unlike undiluted RNAlater, this solution penetrates brain microvasculature resulting in more effective perfusion and RNA preservation. Following perfusion, the brains are then rapidly removed and frozen by immersion in 2-methylbutane in an ethanol/dry ice bath. Brains are chopped with a wire brain slicer or razor before immersion in 2methylbutane in order to obtain consistent blocks that facilitate reproducible sectioning of the tissue. Frozen tissue is stored at 80 -C until sectioning. Twenty-five-micrometer sections are obtained by standard cryostat sectioning at 18 -C, brush-mounted onto PENfoil slides, quickly thaw-mounted and collected in a covered slide box kept inside the cryostat. While thicker sections would reduce the laser dissection time, we found that they are also more likely to be brittle with reduced quality of the morphology, leading to less accurate dissections. PENfoil slides are composed of a rigid

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Fig. 2. Laser microdissection of discrete rat brain sub-regions for microarray gene profiling. Left and middle panels show rat brain sections before and after laser microdissection of the nuclei of interest, respectively; right panels show the location and boundaries of the anatomical regions in question from the Paxinos and Watson atlas [22]. (A) Laser microdissection of dorsolateral and ventral BNST nuclei. f = fornix; Sfi = septofimbrial nucleus; MS = medial septal nucleus; MnPO = medial preoptic nucleus; LSV = lateral septal nucleus; BSTMA = medial anterior BNST; BSTMV = medial ventral BNST; BSTLV = medial lateral BNST; BSTLD = dorsolateral BNST; BSTLJ = juxtacapsular BNST; LGP = lateral globus pallidus. See also Fig. 3A. (B) Laser microdissection of lateral – basolateral amygdala complex. CeM = central amygdala, medial subdivision; CeL = central amygdala, lateral subdivision; BLA = basolateral amygdala; MeAD = medial amygdala, anterior dorsal subdivision; BMA = baso-medial amygdala; DEn = dorsal endopiriform nucleus; VEn = ventral endopiriform nucleus. See also Fig. 3B. (C) Laser microdissection of lateral hypothalamic area. LH = lateral hypothalamus; AHC = central anterior hypothalamus; ZI = zona incerta; PaLM = lateral paraventricular nucleus magnocellular; PaMP = medial paraventricular nucleus parvocellular. See also Fig. 3C. Scale bar = 0.5 cm, as in Fig. 3.

perimeter frame surrounding a very thin transparent membrane insert on which tissue sections are mounted. The insert is composed of polyethylene naphthalate (PEN), which is absorptive in the UV-A range facilitating laser microdissection with UV laser systems. Leica AS LMD uses a UV laser to cut through the tissue and the foil support (Figs. 1D, 2, and 3). We use two alternative dehydration protocols. The first protocol relies on graded ethanol (75%, 75%, 95%, 100%) with sections placed for 20 s in each solution. Sections are then air-dried for 1 min before proceeding to laser microdissection. The main benefit of dehydration in graded ethanol is that it allows for immediate laser dissection following cryosectioning. With the second protocol, the slides are placed in a vacuum desiccator jar containing dry ice in its bottom compartment and desiccated overnight. The next morning, the vacuum is gently released and the slides are taken to sectioning one at the time, while the remaining slides are again stored under vacuum. This latter protocol provides better morphology, greatly improving the ability to recognize brain regions and sub-regions. RNA quality is comparable with the two protocols. For the

dissection of brain regions and sub-regions, we do not stain the tissue because the Leica AS condenser provides sufficient contrast, even with unmounted brain sections, to allow for the recognition of anatomical landmarks (Figs. 2 and 3). The tissue texture is especially evident if the sections are vacuum dessicated overnight as described above. If staining is desired, staining protocols have been devised [8]. 3.2. Laser microdissection Despite initial expectations that the use of laser microdissection systems would expedite dissections [12], the higher anatomical resolution afforded by the currently available devices comes at a cost of time. Therefore, an important aspect in the present protocol is the ability to rapidly and reproducibly laser microdissect the regions of interest with a single pass of the laser, since multiple passes greatly increase the dissection time, thereby reducing RNA quality and making experimental designs with large numbers of animals unfeasible. The laser in the Leica AS LMD can be moved throughout the field of view

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of most of the brain sub-regions commonly dissected in the lab (e.g., the shell and core sub-regions of the nucleus accumbens; the medial, lateral, basolateral, and central amygdala nuclei; the dorsolateral, mediolateral BNST subregions; the subfields of the hippocampus) requires low magnification (4– 5) optics. However, since the laser is delivered through the microscope lens, its power is diminished at lower magnification thus limiting the cutting efficiency at low magnification (4 –5). For this reason, we have tested lenses from various manufacturers and selected a Zeiss 5 Fluar lens, which is engineered for maximum UV transmission, for low-magnification laser microdissection. The use of this lens allows for efficient single-pass cutting at low magnification with greatly reduced dissection time. The microdissected tissue with its membrane support is collected by gravity in the cap of a laboratory tube placed in a rack holder built into the motorized stage. The Paxinos and Watson rat brain atlas and Paxinos and Franklin mouse brain atlas were used for the present study [21,22]. 3.3. RNA extraction, amplification, and labeling

Fig. 3. Laser microdissection of discrete mouse brain sub-regions for microarray gene profiling. Left panels show mouse laser microdissection of the nuclei of interest from mouse brain sections; right panels show the location and boundaries of the anatomical regions in question from the Paxinos and Franklin mouse brain atlas [21]. (A) Laser microdissection of dorsolateral and ventral BNST nuclei. BSTLD = dorsolateral BNST; BSTLJ = juxtacapsular BNST; BSTLP = lateral posterior; BSTLV = ventral lateral BNST; BSTMV = ventral medial BNST; VP = ventral pallidum. See also Fig. 2A. (B) Laser microdissection of lateral – basolateral amygdala complex. The asterisk indicates the lateral – basolateral amygdala complex; the dissection of the medial amygdala is visible on the bottom right (see also Fig. 2B). Cl = claustrum; La = lateral amygdala; BLA = basolateral amygdala; IPAC = interstitial nucleus; AStr = amygdalo-striatal transition; CeMAD = central nucleus of the amygdala antero-dorsal; CeMAV = central nucleus of the amygdala antero-ventral; DEn = dorsal endopiriform nucleus; VEn = ventral endopiriform nucleus. (C). Laser microdissection of lateral hypothalamic area. The asterisk indicates the lateral hypothalamus; the dissected lateral paraventricular nucleus is visible on the right and the dissected medial amygdala is visible on the bottom left (see also Fig. 2C). MGP = medial globus pallidus; AL = ansa lenticularis nucleus; LH = lateral hypothalamus; ZI = zona incerta; PaPo = posterior paraventricular nucleus; MeA = medial amygdala. Scale bar = 0.5 cm, as in Fig. 2.

and its movements are computer controlled. Four tubes can be housed in the stage enabling the dissection of up to four brain regions from multiple slides simultaneously. The operator outlines the desired cut on the microscope field displayed on a computer screen using the mouse pointer. When a satisfactory outline is obtained, the computer is instructed to microdissect the area of interest. The current version of the Leica AS LMD software does not allow one to cut across multiple fields of view under standard computer-controlled operation. Therefore, the dissection

For the extraction of RNA from laser microdissected brain regions, we use the Arcturus Picopure RNA Isolation Kit (Arcturus). Double-stranded cDNA is used as a template for IVT with the RiboAmp OA RNA Amplification Kit (Arcturus) followed by a second round of IVT and biotin labeling using BioArray High Yield RNA Transcript Labeling Kit (Enzo). The Picopure RNA Isolation Kit and the RiboAmp OA RNA Amplification Kit are designed to be used in conjunction with the High Yield RNA Transcript Labeling Kit. We quantify RNA by use of a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies). We use 30 ng of total RNA per sample as starting material for IVT from individual animals without pooling samples from multiple animals. In order to limit dissection time for higher efficiency and RNA quality, we usually dissect only enough tissue to yield approximately 100 –200 ng of total RNA per region of interest, which is sufficient for both microarray analysis and confirmatory RT-PCR. For most of the aforementioned brain regions and sub-regions commonly dissected in the lab, this amount of total RNA can be obtained from about six 25-Am sections from the rat and ten from the mouse. 3.4. Microarray hybridization and analysis Microarrays were hybridized and scanned according to the manufacturer [5]. Hybridization cocktails were boiled at 99 -C, loaded on the RNU34 (Rat Neurobiology U34), RAE230A (Rat Expression 230A), or Rat Genome 230 2.0 arrays and hybridized at 45 -C for 16 h. Washes were performed on the Affymetrix fluidics station or on a custom 24-chip fluidics station using standard wash solutions and stained with a streptavidin – phycoerythrin conjugate

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(Molecular Probes). After staining, chips were scanned with the Affymetrix GeneArray scanner. Following hybridization and scanning, the resulting GeneChip\ files were subjected to three commonly used types of normalization: (1) Global scaling of each chip to a common mean target intensity of 250 using MAS 5.0; (2) normalization to the invariant set of genes across all chips in the experiment using Li and Wong’s PM-only model (dChip) [18]; and (3) background subtraction with quantile normalization using Robust multichip average (RMA) analysis [9,15]. Within-array distributions of expressed genes (Fig. 4) were obtained with GeneSpring 7.1 (Silicon Genetics). M vs. A plots were performed according to Yang and associates [26] to visualize the gene expression differences with the three aforementioned normalization methods (Fig. 5).

4. Results LMD-derived samples subjected to 2 rounds of IVT were compared to samples from manually-dissected brain regions

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subjected to 1 or 2 rounds of IVT (Table 2, Figs. 4 and 5). As shown in Figs. 4A – C, expression values for LMD samples subjected to 2 rounds of IVT and hybridized to RAE230A arrays were normally distributed and highly concordant with manually dissected samples treated in the same manner. Differential gene expression ratios between LMD- and manually-dissected samples subjected to 2 rounds of IVT relative to single amplified samples of the same brain region also hybridized to RAE230A arrays demonstrated a high correlation (r = 0.91) between the double amplified LMD- and manually-dissected samples (Fig. 4D). In Table 2, MAS 5 sample metrics obtained for LMDderived and manually-dissected RNA are shown. Regardless of the GeneChip\ array type used, the percent of genes called Fpresent_ by the MAS 5 detection algorithm was lower and the 3V/5Vratio higher with 2 rounds of IVT than with 1 (Table 2). This is to be expected as the target RNAs prepared with double IVT protocols are shorter (about 200– 1000 bases) than those prepared with a single cycle of IVT, leading to hybridization to fewer features (oligonucleotides)

Fig. 4. Comparison of double amplified laser-microdissected RNA to double and single amplified manually-dissected RNA. (A) Distribution of MAS 5 expression values from the hybridization of Affymetrix RAE230A arrays with target RNA from LMD-derived rat dorsal cingulate cortex (DCG) subjected to 2 rounds of IVT. Expression values were ranked by their normalized intensity (in log scale). (B) Distribution of MAS 5 expression values from the hybridization of Affymetrix RAE230A arrays with target RNA from manually-dissected rat DCG also subjected to 2 rounds of IVT. Both the double amplified samples derived from LMD (A) and the double amplified samples from manual dissection (B) demonstrate a normal distribution. (C) LMD- and manually-dissected samples subjected to 2 rounds of IVT and hybridized to Affymetrix RAE230A arrays also demonstrate normal and very similar distributions when plotted as a histogram in which the logged normalized expression intensity ( y axis) is plotted vs. the number of probe sets (x axis). (D) Scatterplot representing differential gene expression ratios between double and single amplified DCG samples. The log of the ratio of the LMD-derived, double IVT DCG sample to the manuallydissected, single IVT sample of the same region is plotted on the y axis; the log ratio of the manually-dissected double IVT DCG sample to the single IVT of the same RNA is plotted on the x axis. In this plot, deviations from the center along the dashed 45- angle line indicate differences between the single amplified sample and the two double amplified ones. Deviations from the center orthogonally to the dashed line indicate differences between the two double amplified samples. The concentration of the cloud of data points around the center demonstrates a high correlation among the three samples. The slight elongation of the data points along the dashed 45- angle line indicates that the two double amplified samples are slightly more similar to each other than to the single amplified one, consistent with a high correlation (r = 0.91) between the double amplified LMD- and manually-dissected samples.

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Fig. 5. Gene profiling of laser-microdissected brain regions and sub-regions. M vs. A plots of gene expression results using two types of Affymetrix microarrays, rat neurobiology arrays (RNU34) and rat expression arrays (RAE230A). M vs. A plots are representations of the log intensity ratios in which M = log2experimental/control and A = log2SQRT(experimental  control), akin to a 45- rotation of scatterplot [26]. With M vs. A plots, log fold change is represented on the y axis and average absolute log expression level on the x axis [26]. A high linear correlation, as seen in the present results, is indicative of limited differential expression between the two conditions as expected in the brain, in which gene expression changes are typically of limited magnitude and involve a limited number of transcripts [13,17,24,25]. No adjustments were needed to force the distribution of the intensity log ratios to have a median of zero. (A) Gene expression differences between rat prelimbic (PL) and infralimbic (IL) cortices using RNU34 GeneChip\ arrays analyzed with MAS 5.0 software. M = log2(IL/PL); A = log2[SQRT(Mean PL  Mean IL)]. (B) Same as panel A but with dChip (PM-only). (C) Same as panel A but with RMA. (D) Change in gene expression between samples of rat dorsal cingulate cortex in chronic alcohol-treated rats vs. control using RAE230A GeneChip\ arrays analyzed with MAS 5.0 software. M = log2(Chronic/Ctrl); A = log2[SQRT(Mean Chronic  Mean Ctrl)]. (E) Same as panel D but with dChip (PM-only). (F) Same as panel D but with RMA. Probe sets called present or marginal based on the MAS 5 detection call algorithm are represented in blue. Probe sets called absent are displayed in gray. The observation that the vast majority of the genes in the samples analyzed did not show differential gene expression and that the plots scattered symmetrically around the 0 with all of the three normalization methods suggests that the protocol presented did not result in systematic variation that could interfere with the detection of differentially expressed genes.

on the microarray. Comparable increases of 3V/5Vratios were seen in RNU34 (Rat Neurobiology U34) and 230 series arrays, consistent with the fact that the probe sets of the housekeeping genes used for 3V/5Vratios are the same across different generations of Affymetrix arrays. Reductions of genes called Fpresent_ were considerably more pronounced with the older RNU34 array than with RAE230A, although the latter array is much larger and representative of most known brain and peripheral organ transcripts (about 15,000 probe sets are present in the RAE230A vs. about 1500 brain-

specific transcripts in the RNU34) [4]. In fact, over 20% fewer genes were called Fpresent_ with RNU34 arrays hybridized with target RNAs generated by double IVT than with target RNAs generated by single IVT from RNA samples of the same brain region (Table 2). With RAE230A, only about 8% fewer genes were called Fpresent_ with double IVT than with single IVT targets (Table 2). A lower percent Fpresent_ was found with the Rat Genome 230 2.0 than with RAE230A arrays using brain-derived RNA (Table 2), possibly reflecting the larger number of probe sets

Table 2 Comparison of Affymetrix GeneChip\ quality control metrics in LMD- and manually-dissected brain samples Dissection method

Input RNA

RNA amplif. method

Manual LMD

25 Ag 30 ng

Manual LMD LMD a

Probe array type

Total probe sets

% Present

Avg. signal (P)

Avg. signal (A)

Avg. signal (M)

Avg. signal (all)

3V/5V GAPDH

3V/5V h-ACTIN

1 IVT 2 IVT

RNU34 RNU34

1307 1307

47.8% 25.4%

720.6 1637.7

38.7 57.8

89.6 193.2

364.4 461.1

1.31 11.5

1.18 6.7

25 Ag 30 ng

1 IVT 2 IVT

RAE230A RAE230A

15,923 15,923

63.1% 54.9%

491.9 585.2

40.8 34.0

96.8 94.5

326.9 337.4

1.29 13.35

1.46 11.15

30 ng

2 IVT

Rat230 2.0

31,099

43.25%

636.35

74.75

180.32

320.00

2.58

12.49

LMD: laser microdissection; bInput RNA: total RNA used for amplification; cRounds of IVT amplifications used; dScale factor to obtain an average signal intensity of 250; eP = present, M = marginal, A = absent by the MAS 5 detection call algorithm.

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representing ESTs and transcripts from organs other than the brain in the considerably larger Rat Genome 230 2.0 array (about 30,000 probe sets). Experimental data (Fig. 5) were normalized with the Microarray Suite version 5.0 (MAS 5), dChip (PM-only model), and RMA [1,14,18] methods. Although dChip (Figs. 5B, E) and RMA (Figs. 5C, F) displayed considerably less spread at low gene expression levels, as previously observed [15,18], the plots scattered symmetrically around the 0 with all three normalization methods used (Figs. 5A – F). This demonstrates that the vast majority of the genes in the samples, as expected, did not show differential gene expression levels between the two conditions compared. This shows that the laser microdissection, target RNA generation with double IVT, and processing of the microarrays did not result in systematic variation that could interfere with the detection of differentially expressed genes.

5. Discussion The application of transcriptomics and proteomics approaches to accurately dissected anatomically-defined brain regions and sub-regions remains a central focus of current neurobiological investigations as well as a necessary step towards single-neuron neurogenomics and neuroproteomics. The present protocol is designed for simple, rapid, and reproducible laser microdissection of brain regions and sub-regions for gene expression analyses. The emphasis of the protocol is on preserving RNA quality. To this aim, we perfused the animals with an RNA preservative and limited manipulations as much as possible. The texture of the tissue as seen through a Leica AS LMD has sufficient contrast for the recognition of anatomical landmarks used for the identification of brain regions and sub-regions without staining. In order to permit profiling of gene expression changes in complex experimental designs – as is often demanded by behavioral studies – rapidity of execution is ensured by optimization of laser cutting at low magnification. In our lab, we used RNA samples from laser-microdissected tissue subjected to 2 rounds of IVT with three different Affymetrix GeneChip\ arrays: Rat Neurobiology arrays (RNU34), Rat Expression Array 230A (RAE230A), and Rat Genome 230 2.0 arrays. The scant amounts of RNA obtained by laser microdissection require that two rounds of IVT be used for microarray analyses from individual animal samples. The results presented suggest that the newer Affymetrix GeneChip\ microarrays (230 series) are well suited for a double IVT experimental design. In fact, the percent of genes called Fpresent_ with a single IVT amplification protocol in the present report when brain tissue samples were hybridized to RAE230A arrays (Table 2) was in line with the results reported by the microarray manufacturer [7]. The reduction of percent Fpresent_ seen with the two rounds of IVT was more limited with

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RAE230A arrays than with the earlier GeneChip\ arrays tested (RNU34). This reflects the fact that 230 series arrays incorporate improved probe design with fewer probe pairs per gene (11 vs. 16 in earlier arrays) [2,6]. Additionally, 3V representation of probe sequences in the 230 series arrays has been improved based on more comprehensive data on polyadenylation and actual 3V sequence ends [4]. The present results support the increased efficacy of this more 3V targeted microarray design in gene expression profiling of scant RNA samples with two rounds of IVT as required for the analysis of laser-microdissected brain regions and sub-regions.

Acknowledgments Supported by grants DA13821, DA017208 and AA01Q 3191 (PS), VC was partially supported by training grant AA007456 (Neuropsycopharmacology-Multidisciplinary training).

References [1] Affymetrix, Microarray Suite User Guide, http://www.affymetrix. com/support/technical/manuals.affx, 2001. [2] Affymetrix, Rat Expression Set 230, http://www.affymetrix.com/ support/technical/datasheets.affx, 2003. [3] Affymetrix, Standardized Assays and Reagents for GeneChip Expression Analysis, http://www.affymetrix.com/products/expression.shtml, 2003. [4] Affymetrix, Technical Note: Array Design and Performance of the GeneChip Rat Expression Set 230, http://www.affymetrix. com/support/technical/technotesmain.affx, 2003. [5] Affymetrix, Expression Analysis Technical Manual, http://www.affymetrix. com/Auth/support/downloads/manuals/expression_print_manual.zip, 2004. [6] Affymetrix, Rat Genome 230 2.0 Array, http://www.affymetrix.com/ support/technical/datasheets.affx, 2004. [7] Affymetrix, Technical Note: GeneChip\ Expression Platform: Comparison, Evolution, and Performance, http://www.affymetrix. com/support/technical/technotesmain.affx, 2004. [8] Arcturus, Application Note #1. Optimized Protocol for Preparing and Staining LCM Samples from Frozen Tissue and Extraction of High Quality RNA, http://www.arctur.com/, 2004. [9] B.M. Bolstad, R.A. Irizarry, M. Astrand, T.P. Speed, A comparison of normalization methods for high density oligonucleotide array data based on variance and bias, Bioinformatics 19 (2003) 185 – 193. [10] A. Cuello, S. Carson, Micordisection of fresh rat brain tissue slices, in: A. Cuello (Ed.), Brain Microdissection Techniques, John Wiley and Sons, New York, 1983, pp. 37 – 126. [11] J. Eberwine, Single-cell molecular biology, Nat. Neurosci. 4 (2001) 1155 – 1156 (Suppl.). [12] M.R. Emmert-Buck, R.F. Bonner, P.D. Smith, R.F. Chuaqui, Z. Zhuang, S.R. Goldstein, R.A. Weiss, L.A. Liotta, Laser capture microdissection, Science 274 (1996) 998 – 1001. [13] S.J. Evans, P.V. Choudary, M.P. Vawter, J. Li, J.H. Meador-Woodruff, J.F. Lopez, S.M. Burke, R.C. Thompson, R.M. Myers, E.G. Jones, W.E. Bunney, S.J. Watson, H. Akil, DNA microarray analysis of functionally discrete human brain regions reveals divergent transcriptional profiles, Neurobiol. Dis. 14 (2003) 240 – 250. [14] R.A. Irizarry, B.M. Bolstad, F. Collin, L.M. Cope, B. Hobbs, T.P.

74

[15]

[16]

[17]

[18]

[19]

[20]

P.P. Sanna et al. / Brain Research Protocols 15 (2005) 66 – 74 Speed, Summaries of Affymetrix GeneChip probe level data, Nucleic Acids Res. 31 (2003) e15. R.A. Irizarry, B. Hobbs, F. Collin, Y.D. Beaser-Barklay, K.J. Antonellis, U. Scherf, T.P. Speed, Exploration, normalization, and summaries of high density oligonucleotide array probe level data, Biostatistics 4 (2003) 249 – 264. F. Kamme, R. Salunga, J. Yu, D.T. Tran, J. Zhu, L. Luo, A. Bittner, H.Q. Guo, N. Miller, J. Wan, M. Erlander, Single-cell microarray analysis in hippocampus CA1: demonstration and validation of cellular heterogeneity, J. Neurosci. 23 (2003) 3607 – 3615. M.R. Knowles, S. Cervino, H.A. Skynner, S.P. Hunt, C. de Felipe, K. Salim, G. Meneses-Lorente, G. McAllister, P.C. Guest, Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis, Proteomics 3 (2003) 1162 – 1171. C. Li, W. Wong, Model-based analysis of oligonucleotide arrays: model validation, design and standard error application, Genome Biol. 92 (2001) 1 – 11. S.A. Mackler, B.P. Brooks, J.H. Eberwine, Stimulus-induced coordinate changes in mRNA abundance in single postsynaptic hippocampal CA1 neurons, Neuron 9 (1992) 539 – 548. M. Palkovitis, M. Brownstein, Microdissection of brain areas by the

[21] [22] [23] [24]

[25]

[26]

punch technique, in: A. Cuello (Ed.), Brain Microdissection Techniques, John Wiley and Sons, 1983, pp. 1 – 36. G. Paxinos, K. Franklin, The Mouse Brain in Stereotaxic Coordinates, Second ednR, Academic Press, San Diego, 2003, 120os pp. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 4th edition, Academic Press, San Diego, 1998. T. Rabilloud, Proteome Research: Two-dimensional Gel Electrophoresis and Identification Methods, Springer Verlag, New York, 2000, (248 pp.). B. Tabakoff, S.V. Bhave, P.L. Hoffman, Selective breeding, quantitative trait locus analysis, and gene arrays identify candidate genes for complex drug-related behaviors, J. Neurosci. 23 (2003) 4491 – 4498. F.A. Witzmann, J. Li, W.N. Strother, W.J. McBride, L. Hunter, D.W. Crabb, L. Lumeng, T.K. Li, Innate differences in protein expression in the nucleus accumbens and hippocampus of inbred alcohol-preferring and -nonpreferring rats, Proteomics 3 (2003) 1335 – 1344. Y. Yang, S. Dudoit, P. Luu, D. Lin, V. Peng, J. Ngai, T. Speed, Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation, Nucleic Acids Res. 30 (2002) e15.