Microarray, qPCR and in situ hybridization

In a negative control experiment, hybridizing identical dye-swapped samples from the MPIP24k arrays did not return a single statistically significant result. From all MPIP24k and Illumina platform-based microarray experiments, we found over 300 candidate genes which were differentially regulated between HAB and LAB mice in all analyzed brain regions. All data are available in the NCBI GEO database under the accession number GSE29015. From these, the top 32 candidates were investigated in the follow-up qPCR experiment and other analyses (Table S1, excerpt Fig. 1B). In every experiment (that is, throughout all brain regions), ATP-binding cassette, sub-family A member 2 (Abca2), Ctsb, ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5), and tau tubulin kinase 1 (Ttbk1) displayed the largest difference in regulation between lines (>500%). Other genes also attracted our attention, like transthyretin (Ttr) in the BLA and synaptotagmin 4 (Syt4) in the PVN, CeA and BLA. These were oppositely regulated between our two lines, but only in single brain regions. However, we focused on the other genes for this study, as we hypothesized that changes in regulation between most brain regions are more likely to represent general systemic changes, and are therefore more likely to be associated with changes in the phenotypes under consideration. We were able to confirm differences in gene regulation for 13 of the selected genes, by using qPCR on mRNA from the entire brain, with twelve of the candidates delivering statistically significant results (p<0.05; Figure 1A). Although the results for syntaxin 3 (Stx3) showed an opposite effect in the microarray experiments (higher in HAB than in LAB mice) compared to the qPCR, we can rely on the data of the qPCR, as the qPCR data has been replicated a second time with independent samples from the BLA (Fig. 2A). The relatively high number of SNPs detected in the 15^th exon of Stx3 (Table S2) could also explain the higher affinity of Stx3 probes for the HAB-specific mRNA in the microarray experiment. For most genes, the difference in regulation is independent of sex, as the microarray analyses were performed with male mice, and the validation by qPCR was performed with male mice as well as female mice. Thus, for the genes coronin 7 (Coro7), Ctsb, muscleblind-like 1 (Mbnl1), matrix metallopeptidase 15 (Mmp15), Mt1, solute carrier family 25 member 17 (Slc25a17), tribbles homolog 2 (Trib2), zinc finger protein 672 (Zfp672) and Stx3, differential expression could be demonstrated irrespective of sex in HAB vs. LAB mice.

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Figure 1. Gene expression profiles of HAB vs. LAB mice in multiple brain regions.

(A) gene expression confirmed by quantitative PCR and (B) as detected by microarray-based gene expression analysis. N = 6–10 per group. Data are presented as means and for (A) +SEM; T p<0.1, * p<0.05, ** p<0.01, *** p<0.001.

https://doi.org/10.1371/journal.pone.0023604.g001

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Figure 2. Region-specific gene expression in HAB vs. LAB mice.

Expression (A) of Syntaxin3 (Stx3) in the basolateral amygdala (BLA) and (B) of synaptotagmin 4 (Syt4) in the BLA, the central amygdala (CeA) and the hypothalamic paraventricular nucleus (PVN) as measured by quantitative PCR and (C) Syt4 quantified from in situ hybridization. Data are presented as means +SEM; T p<0.1, * p<0.05, ** p<0.01.

https://doi.org/10.1371/journal.pone.0023604.g002

Differential expression of Ttr in the BLA could not be confirmed, in contrast to Syt4, where qPCR (Fig. 2B) and in situ hybridization (Fig. 2C) detected significantly higher expression in the PVN, CeA and BLA of HAB compared to LAB mice.

Sequencing

We used sequencing to identify possible molecular correlates of differences in gene expression between HAB and LAB mice. Analysis of the HAB and LAB Mt1 locus failed to reveal any genetic differences. In contrast, many polymorphic loci were identified at the Ctsb locus. Here, we found 76 SNPs, eight insertions and nine deletions in HAB vs. LAB mice (defining insertion and deletion relative to the mouse strain C57BL/6J). In the promoter region, ten SNPs and two insertions were found. In these ten exons, eight polymorphic sites (all SNPs) were identified (Fig. 3). The vast majority of variations were found in the introns and the downstream enhancer region (Table S3). Interestingly, about six variations are located within a single 230 bp sequence (between −2,269 and −2,045 bp in the promoter). A similar density of polymorphic sites was found in the third and fourth intron, where 18 variations were found in a 550 bp sequence, and 12 additional variations were found in another 400 bp sequence. Additionally, condensation of twelve variable sites could be identified in the downstream enhancer region (at a length of 350 bp).

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Figure 3. Cathepsin B (Ctsb) gene sequence of HAB vs. LAB mice.

Polymorphic sites are shown with positions in the coding sequence referenced to the transcription start in the spliced mRNA in bp. Exons and untranslated regions (UTRs) are indicated by boxes (exons shaded, UTRs completely filled black or white).

https://doi.org/10.1371/journal.pone.0023604.g003

Sequencing of the primer binding sites of the genes we analyzed with qPCR did not reveal any polymorphisms, thus validating the qPCR results. The identified SNPs in the respective sequences are summarized in Table S2. Notably, the ones in Hmgn3 cause amino acid substitutions.

Behavioral testing of KO mice

We could not identify any significant differences in bodyweight between the Ctsb^+/+ wildtype (WT), Ctsb^+/− heterozygous (HET) and their Ctsb^−/− KO littermates, for both male and female mice (data not shown). The average weight difference between male and female mice was 5–6 g.

With respect to locomotion, there were no significant differences between any of the groups, as measured with the EPM (all arm entries, distance traveled) and the open field (OF; distance traveled; Fig. 4A and B). None of the other parameters assessed on the EPM revealed significant differences (data not shown). The data obtained from the TST were not suitable for analysis – all groups showed climbing behavior, so an evaluation regarding immobility parameters was not possible. In the OF, the different groups of male mice failed to show significant differences in the anxiety parameters (Fig. 4C), but female Ctsb KO mice showed a significantly decreased inner vs. outer zone path length ratio (Fig. 4D), combined with a decreased time spent with path length in the inner zone of the test field, reaching a statistical trend (Kruskal-Wallis H test – KWH: p<0.1, corrected Mann-Whitney U test – MWU: p<0.1 only for the WT vs. KO comparison; mean time spent in the inner zone ± SEM for KO: 7.6±1.0 s, HET: 11.5±1.2 s, WT: 13.0±1.7 s; mean path traveled in the inner zone ± SEM for KO: 1.2±0.2 m, HET: 1.8±0.2 m, WT: 1.9±0.2 m).

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Figure 4. Phenotypes of Cathepsin B (Ctsb) knockout (KO) vs. heterozygous (HET) and wildtype (WT) mice.

(A) Locomotion of male and (B) female mice in the open field, (C) anxiety-related behavior of male and (D) female mice as measured in the open field; (E) depression-like behavior of male and (F) female mice as reflected by the forced swim test. Data are presented as means +SEM; * p<0.05.

https://doi.org/10.1371/journal.pone.0023604.g004

In male mice, a trend towards decreased exploratory drive, increased self-grooming and increased arousal was observed on the elevated platform (EPF), as also indicated by the increased gut activity in the Ctsb KO males compared to wildtype mice (KWH: p<0.1; mean grooming frequency±SEM for KO: 1.6±0.3, HET: 2.7±0.5, WT: 1.4±0.5; mean frequency of exploratory head-dips±SEM for KO: 30.8±2.8, HET: 43.1±3.6, WT: 35.8±1.9; mean frequency of defecation±SEM for KO: 4.4±0.6, HET: 3.1±0.5, WT: 2.5±0.5). Female mice did not show any significant difference.

Also, no differences were observed after stress exposure in the stress-reactivity test (SRT; data not shown).

Although no significant differences could be observed between WT and KO male mice (Fig. 4E) in the FST, several parameters showed significant differences when comparing female mice. Reduced swimming time indicated a less active coping strategy of Ctsb KO mice (Fig. 4F). A statistical trend was observed for the time spent floating in the same direction (KWH: p<0.1, corrected MWU: p<0.1 for the WT vs. KO and HET vs. WT comparisons; mean time floating±SEM for KO: 244±6.2s, HET: 241±4.8 s, WT: 215±11.1 s).

Animals and behavioral tests

All animals were kept in the animal facility of the Max Planck Institute of Psychiatry, under standard housing conditions (room temperature 23±2°C, relative air humidity 60±5%, 12 h/12 h dark/light cycle with lights on at 7 am). Animals were kept in groups of two to four per cage, with light levels not exceeding 100 Lux at any time. Mice from each line were selected from generations G16-32, based on their anxiety-related behavior, as measured by their performance on the EPM. The animals were also tested with the TST [15] for 6 minutes to assess their depression-like coping strategy. Through heterozygous breeding, we created litters of mice containing both Ctsb deficient animals and control (non-deficient) animals (sequences of genotyping primers are available upon request). 42 male and 55 female mice Ctsb KO, HET and their WT littermates [46], [65] were tested for locomotion and anxiety-related behavior in the EPM and OF tests for 5 minutes, for depression-like behavior in the FST and TST for 6 minutes each [15], and for stress hormone response to a 15 minute SRT as described by Touma et al. [60]. The OF test we used had illumination of 10 Lux in the central part of the field. To assess explorative behavior, an EPF test [66] was performed for 5 minutes. The apparatus we used for this test consisted of a centered platform of grey polyvinylchloride 40 cm high and 10 cm in diameter, mounted to a wooden plate (42×42 cm) and surrounded by three wooden walls (w×h: 42×58 cm), and left open at the front for observation. The metrics we assessed on the EPF test were the frequency and duration of rearings, the delay before the first rearing, and the frequency of exploratory head-dips over the platform edge and the frequency of grooming behavior, which are indicative of displacement activity.

Testing was started on the Ctsb KO mice and their littermates at the age of eight weeks, starting with the EPM test on day 1, and continuing to TST on day 3, OF on day 7, EPF on day 9, SRT on day 11, and FST on day 14. All of the behavioral tests were digitally recorded or video-taped. The results of the EPM test, FST and TST were analyzed as in previous studies [15], [16]. The same evaluation scheme was applied to the EPF test, by counting the frequency of head-dips when mice reached the platform's edge with their shoulder line, the frequency, latency and duration of rearing, and the frequency of grooming behavior. The OF tests were analyzed using Anymaze v. 4.71 (Stoelting&Co., Wood Dale, IL). The order in which the mice were tested was random, and evaluation was performed by a trained observer who was not aware of the lines of the mice. All chemicals and reagents not declared otherwise were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Ethics statement

The government of Upper Bavaria (Oberbayern) approved our animal experiments (approvals IDs – AZ: 55.2-1-54-2531-64-07 and 55.2-1-54-2531-73-02), which were conducted according to the current regulations for animal experimentation in Germany and the European Union (European Communities Council Directive 86/609/EEC).

Tissue dissection by micropunching

Animals were anesthetized and killed by decapitation at the age of ten weeks, three days after the TST. For MPIP24k gene expression analysis, six male HAB and LAB mice from breeding generation 16 were used, while for subsequent qPCR analysis, we used eight female HAB, LAB and CD1 mice from generation 22. A further group of eight male mice from each line, from generation 29, was used for micropuncture. The brains of the mice were collected, dissected into 200 µm slices, and mounted from rostral to caudal to Superfrost microscope slides (Menzel, Braunschweig, Germany) in a cryostat (Microm MH50, Microm, Walldorf, Germany). The brain areas of interest were acquired from these slices by micropuncture, through a method described in previous studies [67], utilizing punchers with a diameter of 0.5 and 1 mm (Fine Science Tools, Heidelberg, Germany). The brain regions collected included the Cg, the NAc core and shell, the PVN, the SON, and the BLA, CeA and medial amygdala (MeA). Punches of 1 mm diameter were collected from bregma +1.3 mm to +0.9 mm, twice sampling the tissue medially about 0.5 mm from the dorsal tissue border to receive the Cg, and bilaterally sampling the NAc core, which inevitably included a minor part of its shell around the anterior commissure. Further tissue was collected medially 0.8 mm above the ventral tissue limit (Ø = 1 mm), and bilateral-dorsolaterally from the optic tract (Ø = 0.5 mm), to acquire tissue from the PVN and SON, from bregma −0.56 mm to −0.96 mm. Amygdala tissue samples were collected bilaterally from two slides for each region with 1 mm diameter punches. CeA was collected from bregma −0.96 mm to −1.36 mm dorsomedially from the ventral end of the external capsule, MeA from −1.16 mm to −1.56 mm dorsolaterally from the optic tract, and BLA/LA was collected from bregma −1.36 mm to −1.76 mm from in between the bifurcation of the external capsule. All coordinates were based on the Mouse Brain Atlas [68].

Tissue laser microdissection

Six male HAB, six male NAB, and six male LAB animals from generation 25 were sacrificed using the previous procedure, except for obtaining 25 µm slices in the cryostat (Microm). Only brain slices containing the brain areas of interest were sampled, including the anterior part of the Cg, the PVN, the SON, the anterior DG, the CeA, and the BLA. For the DG, the same coordinates as for the PVN and SON were used. To cover the whole area of interest, an overall depth of 400 µm per region was chosen. After sampling the Cg, BLA and CeA brain regions, four brain slices were mounted onto an LMD6000 metallic frame slide, which was covered with a membrane of polyethylene terephthalate (Leica Microsystems Deutschland, Bensheim, Germany), while the following four slices were mounted onto Superfrost slides (Menzel). This procedure was repeated for the next eight slices. For the region containing the PVN, SON and DG, only the first two slices were mounted to the LMD6000 frame, with the next two mounted to the Superfrost slides, and so on, again alternating between them. Only the LMD6000 slides were used for further processing, and these slides were stored at −80°C. Before we performed laser-microdissection, the brain slices on the LMD6000 frame slides were stained with cresyl violet. Our staining protocol started with staining the slides for 90 seconds in cresyl violet, followed by washing them in 70% and 96% ethanol for 20 seconds each, and finally washing them in isopropanol for 5 minutes. The slides were refrozen after staining, and were then processed with the laser-microdissection microscope (AS LMD, Leica). For dissecting these brain areas, we chose a magnification of 100×, laser power between 80–100%, and speed varying between 1 and 4. Cut-out brain areas were stored in the caps of 0.2 ml PCR soft tubes (Biozym Scientific, Hessisch Oldendorf, Germany), and were cooled with dry ice immediately after the completion of one brain region.

Total RNA isolation and amplification

Total RNA was extracted in presterilized 1.5 ml safelock tubes (Eppendorf, Hamburg, Germany), using a standard TRIzol (Invitrogen, Karlsruhe, Germany) chloroform protocol. After the sample tissue was homogenized by adding 300 µl TRIzol with a pipette, 1 µl linear acrylamide (5 mg/ml, Ambion, Austin, TX) and 60 µl chloroform (Carl Roth, Karlsruhe, Germany) were added, and the samples were vortexed. This was followed by centrifugation for 5 min at 18°C and 13,000 rpm. The RNA was then precipitated by applying 180 µl isopropanol (Carl Roth) overnight at −20°C, centrifuging the sample at 4°C and 13,000 rpm for 30 min, and then washing it twice in 500 µl 70% ethanol (Carl Roth), with ten-minute centrifugation steps at 4°C and 13,000 rpm in between. After the last centrifugation step, all remaining liquid was removed with a pipette, and the pellets were dried in an incubator for 15 min at 45°C and stored solved in 13 µl of water (Ampuwa, Braun Melsungen, Melsungen, Germany).

For the MPIP24k experiment, the total RNA we extracted was amplified and dye-coupled in two rounds, using Ambion's Amino Allyl MessageAmp aRNA kit (Ambion). T7 oligo(dT) primers were used for the first round of reverse transcription to select specifically for mRNA, and random hexamer primers were used for the second round of reverse transcription.

For the Illumina MouseWG-6 experiment, the RNA was amplified using the Illumina TotalPrep RNA Amplification kit (Ambion). 5 µg of material per sample was required for loading the microarray slides. We ensured correct quantification by measuring optic density in a NanoPhotometer (Implen, Munich, Germany), and through additional analysis on agarose gel. Samples not fulfilling all of our homogeneity criteria (samples with inadequate concentrations or size distribution) were excluded from further analysis.

Hybridization and quantification of the MPIP24k arrays

Ten array slides per brain region (MPIP24k arrays containing 24,192 probes, MPI of Psychiatry, Munich, Germany [69], [70], platform available in NCBI/GEO with accession number: GPL7467; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL7467) – serving as technical replicates - were prehybridized prior to the experiment in a prehybridization buffer, for 1 h at 42°C. The buffer consisted of 125 ml formamide, 62.5 ml 20×SSC, 2.5 ml 10% SDS, 2.5 ml BSA at 10 mg/ml and 57.5 ml of water. The array slides were then washed in water and isopropanol, and dried by three minutes of centrifugation (Megafuge 1.0R, Heraeus, Hanau, Germany) in 50 ml tubes at 1500 g (Sarstedt). Dye-coupled aRNA samples were mixed with the opposite dye-coupled samples of the relevant regions from the other mouse line, and were loaded with a hybridization buffer (consisting of 500 µl formamide, 250 µl 20×SSC, 10 µl 10% SDS, 5 µl mouse-COT1-DNA at 20 mg/ml (Invitrogen) and 40 µl poly adenylic acid at 2.5 µg/µl (Amersham Biosciences) to five arrays each under m-Series LifterSlips (Menzel). All arrays were hybridized at 50°C for 16–17 h in separate hybridization chambers. The arrays were then washed in 2×SSC and 0.1% SDS for 5 minutes at 42°C, for 10 min at room temperature in 0.1×SSC and 0.1% SDS, four times in 0.1×SSC for 1 min each, and finally for 20 s in 0.01×SSC solutions. The slides were dried by three minutes of centrifugation at 1,500 rpm. Then, the arrays were scanned on a PerkinElmer ScanArray 4000 (PerkinElmer Life and Analytical Sciences, Shelton, CT) laser scanner, using automatic focusing and laser power between 60 and 80 for Cy3, and 40 and 70 for Cy5. This ensured that, on average, the same fluorescence intensities were reached in both dyes, and that not more than 1–2% of spots had fluorescence intensities above the saturation point. Quantification of all array data was performed with QuantArray software (GSI Lumonics, Billerica, MA), by applying a fixed-circle quantification protocol, with manual positioning of all grids over the hybridized spots. To provide a negative control for the hybridization and evaluation procedure, excess aRNA was used for an additional hybridization in both Cy3 and Cy5 combinations.

Hybridization and quantification of the Illumina MouseWG-6 arrays

The platform description is available at NCBI GEO, accession Number GPL4234 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=fjqlzekgyecycng&acc=GPL4234). Each microarray slide had the capacity for six samples. Samples containing the same brain regions were hybridized in the same batch, so a maximum of six individual mice of each breeding line were compared per batch. Reagents and material for the analysis were provided by Illumina. In brief, each sample was mixed with hybridization buffer and loaded onto the designated array field, and the slides were then put into hybridization chambers and incubated for 17 h in Illumina incubation chambers. The arrays were washed in several steps, incubated with Cy3-Streptavidin, again washed several times, and dried by centrifugation. Finally, data on fluorescence was gathered using a BeadStation scanner (Illumina), and analyzed with the BeadStudio (Illumina) software. The manufacturer's built-in controls were also analyzed, including hybridization controls and sample dependent parameters. Only microarrays meeting Illumina's recommended quality control criteria were used for further evaluation.

Quantitative PCR (qPCR)

Candidate genes were selected for further analysis if they showed at least 40% greater regulation in one region, and at least 30% greater regulation in all other regions, in HAB mice relative to LAB mice, with adjusted p-value<0.05.

Three 200 µm coronal sections were also taken at different brain levels, for representative whole brain analyses. A maximum of 1 µg of total RNA was reverse transcribed with Superscript II (Invitrogen, Karlsruhe, Germany) after DNAse treatment. For quality control, a small aliquot of each cDNA was analyzed on an agarose gel. cDNA of male or female HAB or LAB mice was analyzed by qPCR, using the QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany). The oligonucleotide primers were designed based on the Primer3 algorithm [71], and were then purchased from Sigma-Aldrich. We selected gene products for quantification by qPCR if they showed: significant expression differences between HAB and LAB mice with an adjusted p-value of <0.10, at least 30% greater regulation between lines, and differential expression in the microarray experiment in all, or at least three, analyzed regions. All the primers we used for qPCR are listed in Table S1. Experiments were performed in duplicates on the Lightcycler®2.0 instrument (Roche Diagnostics, Mannheim, Germany) using the following PCR process: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation (95°C for 10 s), and then a combined annealing and extension phase (60°C for 30 s). At the end of every run, a melting curve (50–95°C with 0.1°C/s) was used to ensure the quality of the PCR product. Crossing points (Cp) were calculated with LightCycler® 4.0 software (Roche Diagnostics), using the absolute quantification fit points method. The threshold and noise band were set to the same level in all compared runs. Relative gene expression was determined by the 2^−ΔΔCT method [72]. Cp were normalized to the housekeeping genes Gapdh, Hprt1, Atp2b1, Rpl13a and Polr2b, or any combination of two of the mentioned genes. Fold regulation values were calculated relative to the expression mean of the group displaying the lowest expression.

In situ hybridization (ISH)

ISH using ³⁵S-UTP labeled ribonucleotide probes was performed as described in previous studies [73], [74] to detect Syt4 mRNA. Briefly, sets of sections for each riboprobe ISH were fixed in 4% paraformaldehyde, and acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine/HCl. Afterwards, the slides were dehydrated with progressively higher ethanol concentrations, degreased with chloroform, rinsed in ethanol and then air dried. The antisense cRNA probe for Syt4 (470 bp) was transcribed from a linearized plasmid, and labeled using SP6 polymerase transcription systems, using a standard labeling reaction mixture consisting of 1.5 µg of linearized plasmid, 1× transcription buffer, 0.12mCi of ³⁵S-UTP, 1 mM dNTPs, 16.7 mM DTT, 40 U of RNAse inhibitor, and 20 U of the polymerase. The reaction mix was incubated at 37°C for three hours, and the labeled probe was then separated from free nucleotides with spin columns (Qiagen, [74]). The tissue sections (5 sections per slide) were saturated with 100 µl of hybridization buffer (Tris HCl, EDTA, NaCl, formamide, 5 M DTT, Dehnhard's solution, DEPC H₂0, 50% dextran sulfate) containing 106 cpm ³⁵S labeled riboprobe. Brain sections were coverslipped and incubated in humid chambers for 18–22 h, at a temperature of 55°C. After this was done, the slides were rinsed in 2×SSC, treated with RNAse A (20 mg/l), washed in progressively less concentrated SSC solutions, dehydrated by progressively more concentrated ethanol solutions, and air-dried, before they were photographed with Kodak BioMax MR films (Amersham, Braunschweig, Germany) over a 14-day period. Afterwards, the films were fixed, developed, and digitized, and the radiation-induced blackening of different brain regions was quantified by image analysis, using Scion Image (Version 4.0.3.2, Scion Corporation, Frederick, MD) software. Autoradiograms were analyzed by computer assisted optical density readings (relative grey intensity as a measure of relative expression) of the respective area, as well as the relative size of the labeled area. Three to six brain sections of each individual were quantified by an observer unaware of the individual's breeding line, and the highest expression (hybridization signal of a certain region, minus the background signal of a nearby structure that does not express the gene of interest) was used to calculate each mRNA expression, respectively.

Sequencing of candidate genes

To identify polymorphic sites between the HAB and LAB mice, the Ctsb and metallothionein 1 (Mt1) coding genes were sequenced, as these were the two candidate genes whose involvement in diverse molecular pathways was best described. In both cases, about 2,500 bp of the gene promoter, all exons and introns (if the latter did not exceed 2,000 bp), and about 2,000 bp of the downstream enhancer regions (DER) were sequenced. Sequencing primers were designed to cover 500 to 600 bp.

Sequencing was also used to verify qPCR reaction products, and their primer binding sites. In this case, the fragments used were between 200 and 500 bp.

DNA was isolated from tail tips using the NucleoSpin Tissue (Macherey-Nagel) kit, from fresh fecal samples as described by Murgatroyd et al. [75], or sequencing was prepared based on cDNA. PCR reactions were set up in three HAB and three LAB samples. The primers we used are summarized in Table S4. All amplifications were carried out using Taq polymerase (Fermentas, St. Leon-Rot, Germany) in 25-µl reactions with the following procedure: initial denaturation at 94°C for 4 mins, 40 cycles of denaturation (94°C for 1 min each), annealing (52–66°C for 1 min) and extension (72°C for 1 min), followed by 10 min of final extension at 72°C. 15–20 µl of PCR or qPCR product were purified by washing the sample twice with nuclease-free water, using the NucleoFast 96 PCR Clean-up kit (Macherey-Nagel). Clean-up plates were centrifuged at 9°C and 4,500 g for 10 min (Heraeus Multifuge 4KR, Thermo Fisher Scientific, Waltham, MA), and resolved for ten more minutes in 25 µl of water on a shaker (Eppendorf). 2.4 µl of cleaned up PCR product was used for the sequencing reaction (BigDye Terminator Kit, Applied Biosystems, Foster City, CA), with 1.2 µl of sequencing buffer, 0.4 µl BigDye reagent and 1 µl of the forward primer per sample. If sequencing reaction results were unclear or unreadable, the sequencing reaction was also performed with the reverse primer. Sequencing reactions were set up on ThermoFast 96 PCR plates (ABgene, Hamburg, Germany) using a PTC-225 Gradient MultiCycler (MJ Research, Miami, FL) using the following procedure: initial denaturation at 96°C for 1 min, 35 cycles of denaturation (96°C for 10 s), annealing (50°C for 5 s), and finally extension (60°C for 4 min). Sequencing reaction products were purified by washing them twice in 20 µl injection solution, using Montage SEQ96 plates (Millipore, Billerica, MA) on a vacuum pump (Biomek 2000 Laboratory Automation Workstation, Beckman Coulter, Fullerton, CA), and the reaction products were then transferred to 96 well plates. Sequences were determined by capillary electrophoresis on a 3730 DNA Analyzer (Applied Biosystems) at the HelmholtzZentrum's Institute of Human Genetics (Neuherberg, Germany). Sequence analysis and comparison were done using FinchTV Ver. 1.2 (Geospiza, Seattle, WA) and BioEdit Ver. 7.0.2 (Tom Hall, Ibis Biosciences, Carlsbad, CA) softwares.

Statistical analysis

All data, except for data from high throughput gene expression profiling, was analyzed using SPSS Ver. 16.0.1 (Chicago, IL). The Kruskal-Wallis test (KWH) was used for comparisons over more than two groups, with subsequent Mann-Whitney tests (MWU), as well as sequential Bonferroni correction for multiple testing (if applicable).

For statistical evaluation of the MPIP24k arrays, analytic methods were applied as described in previous studies [76], [77]. In brief, an MA-plot was first generated to display the raw fluorescence intensities of Cy5 (R) and Cy3 (G), with M = log₂ R/G and A = log₂ . The data were then normalized to exclude systematic and technical errors. The data were first normalized twice, by subtracting a function c from the logarithms of the fluorescence intensities (log₂ R and log₂ G). First, a global normalization was performed, based on the assumption that R and G correlate:In the second normalization step, an intensity-dependent normalization was added, which was done by applying a LOESS smooth operation, as described in the R software package (http://www.r-project.org). This was calculated with:where c(A) stands for the LOESS smooth of the MA-plot. 40% of the values in the MA-plot were used to calculate the LOESS smooth. In several subsequent normalization steps, we minimized differences that resulted from an unequal distribution of probes in the array production, or from unbalanced fluorescence intensities within one array slide. All data were then merged into a matrix. P-values for multiple testing were calculated by permutation, and are therefore called adjusted p-values.

For the Illumina Mouse WG arrays, Illumina BeadStudio gene expression results were analyzed using a statistical procedure similar to the one for the MPIP24k experiment. All analyses were performed using R-packages, based on ‘beadarray’ described by Dunning et al. [78], which simplifies comparisons between high numbers of arrays. First, pair-wise box plots were generated to compare the mean amount of expression within each line and brain region. Normalization for expression values was applied to all samples with the ‘QSpline’ function. Clustering using the ‘hclust’ function shows that each brain region per line shows similar expression patterns. Three samples from different brain regions were identified as inadequate during the scan process, and have therefore been excluded from further analysis. For differential expression analysis, the functions of the ‘limma’ package were applied to log₂-transformed values. The resulting matrix has been used for all subsequent analyses. Significantly regulated genes were ranked using an empirical BAYES method, as implemented in the limma R-package [79], [80].