Mouse breeding, tissue preparation and total RNA isolation
We obtained CBA/CaOlaHsd (CBA), BALB/cOlaHsd (BALB), C57Bl/6JOlaHsd (BL6), DBA/2OlaHsd (DBA), and C57Bl/10ScSnOlaHsd (BL10) mice from Harland Laboratories, and C57Bl/10ScSn-Dmdmdx/J (mdx) mice from Jackson Laboratory at the age of 6 weeks. Mice were kept under standard conditions and were sacrificed by cervical dislocation when 8 weeks old. Hindlimb muscles (m. quadriceps femoris) were dissected and promptly snap-frozen in isopentane at -80°C. Total RNA was prepared by disrupting tissue using mortar and pestle and subsequent homogenisation by a rotor-stator homogenizor (Ultra-Turrax T25, Janke & Kunkel IKA-Labortechnik) in RNA-Bee (Campro Scientific) until uniformly homogenous (15–45 sec). Total RNA was isolated according to manufacturer's instructions followed by purification using RN-easy columns (Qiagen). Quality and yield was determined using Lab-on-a-chip (BioAnalyzer, Agilent).
Target preparation and hybridisation
Aminoallyl labelled cDNA (aa-cDNA) was prepared based on a previously described protocol[14]. Aliquots of 1 μg of total RNA in the presence of 2 μg amino-TN6 primer (5'-NH2-(CH2)6-TN6, Eurogentec) were adjusted to a volume of 21 μl with DEPC-treated H2O (diethyl pyrocarbonate, Sigma), heated for 10 minutes at 70°C and chilled on ice for 10 minutes. Reverse transcription mastermix (1.8 μl RevertAid RNaseH-M-MuLV reverse transcriptase (200 U/μl, MBI Fermentas), 6 μl 5x first-strand buffer (MBI Fermentas), and 1.2 μl 25x aa-dUTP / dNTP solution (2 μl 50 mM dATP, 2 μl 50 mM dCTP, 2 μl 50 mM dGTP, 1.2 μl 50 mM dTTP, 0.8 μl 50 mM aminoallyl-dUTP (Ambion)) was added per reaction and incubated at room temperature for 10 minutes followed by 2 hours at 42°C. RNA was hydrolysed by addition of 10 μl 0.5 M EDTA and 10 μl 1 M NaOH and incubation at 65°C for 30 minutes followed by neutralization by addition of 10 μl 1 M HCl. Aminoallyl labelled cDNA was then purified by combining 300 μl of PB-buffer (Qiagen) to 60 μl of the neutralized sample and centrifuged through a Qiaquick column (Qiagen) at 13000 rpm for 1 minute. Two washing steps were performed by spinning 500 μl of 75% EtOH at 13000 rpm for 1 minute while discarding the flow-through. To remove ethanol-traces the columns were centrifuged for an additional minute. cDNA was recovered by eluting three times using 30 μl basic H2O (3.3 mM NaHCO3 buffer, pH 9.0) and concentrated to a volume of 6.66 μl using a speedvac. Aliquots of Cy3 and Cy5 reactive dyes (PA23001, PA25001, Amersham) were prepared by dissolving each vial of monoreactive dye in 40 μl fresh anhydrous DMSO (Sigma) and dividing into aliquots of 2 μl followed by vaccuumdrying until dry and subsequent storage at 4°C in the presence of silica. Fluorescent dyes were coupled by adding 3.33 μl of bicarbonate buffer (1 M NaHCO3 buffer, pH 9.0) to the aa-cDNA sample and dissolving the dried aliquot of reactive dye, followed by incubation at room temperature for 1 hour in the dark. To the samples 4.5 μl 4 M hydroxylamine (Sigma) was added and incubated at room temperature in the dark for 15 minutes, followed by addition of 186 μl TE-3-buffer. Hybridisation mixtures were prepared by combining a Cy3-labeled cDNA sample with a Cy5-labeled cDNA sample and 10 μl Mouse-Hybloc (1 μg/μl, Applied Genetics Laboratories) followed by removing uncoupled dyes by spinning through a pre-wetted Microcon column (YM30, Amicon) for 8 minutes at 13000 rpm. Hybridisation mixture was washed by spinning 500 μl TE-3-buffer through the column and discarding the flow-through. This step was repeated two times as 2 μl yeast-tRNA (10 μg/μl, Sigma) and 2 μl polyA-RNA (10 μg/μl, Sigma) were added during the last step. Mixture was collected by inverting the column and spinning for 1 minute at 13000 rpm. Hybridisation mixture was finalized by adding TE-3-buffer to 84 μl together with 17 μl 20x SSC and 3 μl 10% SDS followed by denaturing at 100°C for 2 minutes, renaturing at room temperature for 15 minutes and spinning at 13000 rpm for 10 minutes. Labelled target was hybridised overnight on murine oligonucleotide microarrays (65-mer with 5'-hexylaminolinker, Sigma-Genosys mouse 7.5 K oligonucleotide library, spotted in duplicate). Hybridisation occurred in a automatic hybridisation station (GeneTac, Perkin Elmer) and was followed by washing with 5x 2xSSC + 0.1% SDS at 30°C, 5x 1xSSC at 30°C, 3x 0.2xSSC at 30°C, 1x 0.2xSSC at 65°C, 2x 0.2xSSC at 30°C, and subsequently scanned as described previously[15].
Experimental design, data extraction and analysis
Gene expression profiles from hindlimb muscle derived from 2 male animals of each strain were generated using dye-swap experiments. Subsequent duplicate spots on each array resulted in 8 replicate measurements per gene. Targets were assigned at random to the arrays, while avoiding co-hybridisation of samples from the same strain. GenePix Pro 3.0 (Axon) was used for feature extraction and quantification. Genes were considered as being expressed when the corresponding feature was not flagged by the algorithm provided by GenePix. Local background corrected spot intensities were normalized using Variance Stabilization and Normalization (VSN) in R [16]. Array data has been made available through the GEO data repository of the National Center for Biotechnology Information under series GSE662. Correlation between individuals was calculated using Pearson's correlation coefficient. Significantly differential expression levels were determined using MA-ANOVA (MAANOVA2.0 The Jackson Laboratory http://www.jax.org/staff/churchill/labsite/software/anova/), hierarchical t-test [11] and the False Discovery Rate [17] selection procedure.
Quantitative Reverse Transcription Polymerase Chain Reaction
qPCR was performed in duplicate for each individual resulting in four measurements per strain per gene. cDNA was prepared by reverse transcription using 1 μg total RNA as template. Random hexamers (40 ng) were used to prime the transcription after heating 10 minutes at 70°C followed by chilling on ice for 10 minutes. cDNA was synthesized by RevertAid RNaseH- MuLV reverse transcriptase and accompanying buffer (MBI-Fermentas) using 1 mM dNTPs. The mixture was incubated at room temperature for 10 minutes before a 2 hour incubation step at 42°C, followed by 10 minutes at 70°C. Quantitative PCR was performed using the Lightcycler (Roche). PCR mixture was prepared by combining cDNA dilution, 10 pmol forward and reverse primer, MgCl2 (4 mM) with 4x home-made LC mastermix (0.9 mM dNTPs, BSA (1 μl/μl, Pharmacia Biotech), Taq polymerase (0.8 U/μl), 4x SYBR Green I (Molecular Probes), 4x AmpliTaq Reaction Buffer (Perkin Elmer)) to a total volume of 20 μl. Amplicons were generated during 45 cycles with annealing temperature set at 55°C. Optimal cDNA dilutions and relative concentrations were determined using a dilution series per gene. Replicate experiments (n = 4) were normalized to 1 and relative expression values were determined by calculating the ratio per gene over the average relative expression of genes, which show no differential expression on both microarray and qPCR (dysferlin, cystatin B, and thrombospondin 4). Significance levels were calculated with a one-sample t-test. PCR primer pairs were designed using the Primer3 search engine, available at: Primer3 Software Distribution http://frodo.wi.mit.edu/primer3/primer3_code.html. The screened genes and the oligonucleotide primer pairs used for each of the genes in this study correspond to the following nucleotides: myomesin1, 4761–4780 and 4865–4884 (NM_010867); tropomodulin1, 670–689 and 878–897 (NM_021883); dysferlin, 4218–4237 and 4353–4372 (AF188290); cystatinB, 3–22 and 151–170 (NM_007793); thrombospondin4, 2167–2186 and 2289–2308 (NM_011582).
