strain: CD1 age: adult; 8weeks gender: male tissue: extensor digitorum longus (EDL) muscle transfected with: none (control) tissue subtype: single skeletal muscle fibre time point: 7 days after transfection molecule subtype: long RNA (>200 bp)
EDL muscles were isolated through a small hindlimb incision and 10^11 AAVs particles were injected along the muscle length. Muscles were analyzed 7 and 14 days post-injection.
Male CD1 mice were fed ad libitum (two mice per cage). Eight weeks old mice were used for EDL infection with AAVs.
Mice were sacrificed by rapid cervical dislocation and wild-type or AAV injected EDL muscles were collected. Muscles were incubated for 45 min. at 37°C in 1 ml high-glucose Dulbecco’s modified Eagle medium (DMEM; Life Technologies) containing 10 mg type I collagenase (220 U mg-1; Sigma-Aldrich). Collagenase-treated muscles were sequentially rinsed for 2 min. in DMEM, DMEM supplemented with 10% fetal bovine serum (FBS), and transferred in DMEM again. Single myofibers were liberated by gentle physical trituration with a wide-mouth plastic Pasteur pipette, and the triturating process was repeated several times until the mayor part of myofibers were dissociated. Intact, no contracted and well-isolated myofibers were picked under stereo-microscope and washed in phosphate buffer saline (PBS). ZsGreen-expressing myofibers of AAV injected EDL muscles were selected using an inverted microscope (DMI4000, Leica). All myofibers were collected within 45 min. from the last trituration step. Each single isolated myofiber was lysed in 250 µl of TRIzol Reagent (Life Technologies) and RNA was extracted in the aqueous phase following the manufacturer’s instructions. To purify RNA, spin-columns of the RNeasy Micro Kit (Qiagen) were used, adding 70% ethanol to the aqueous phase and following the Qiagen protocol.
Before preforming microarray experiments about 1/5 of the purified RNA was used to test, by qRT-PCR, the MCU overexpression or downregulation in the selected myofibers. RNA was retrotranscibed using the SuperScript III Reverse Transcriptase (Life Technologies) according to the manufacturer’s specifications. Gene-specific primers for exogenous MCU (AAV MCU), endogenous MCU, and ZsGreen were selected with Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and the specificity of each primer set was monitored by dissociation curve analysis. Tioredoxin 1 was chosen as reference gene. qRT-PCR experiments were performed in a 7500 Real-Time PCR System (Life Technologies) using the SYBR Green technology of GoTaq qPCR Master Mix (Promega). The remaining purified RNA from myofibers demonstrating MCU reduction or overexpression was exponentially amplified using the TransPlex WholeTranscriptome Amplification 2 Kit (Sigma-Aldrich) according to Chemello F. et al., PlosOne 2011 to obtain a sufficient amount of cDNA for microarray experiments. Briefly, RNA was reverse transcribed in a cDNA library, and then library was exponentially amplified for 18 cycles, few cycles below the amplification “plateau” observed in a PCR test reaction. To remove the residual primers and nucleotides, amplification product was purified with the GenElute PCR Clean-up columns (Sigma-Aldrich). Resulting cDNA has been quantified with Nanodrop ND-1000 spectrophotometer (Thermo Scientific). 2 µg of amplified-purified cDNA were direct labeled using the Genomic DNA Enzymatic Labeling Kit (Agilent Technologies) as described in the manual. The kit uses random primers and the exo-Klenow fragment to directly label cDNA samples with Cy3-dUTP nucleotides. Labeled cDNA was purified using the Amicon 30kDa filters (Millipore) and quantified using NanoDrop ND-1000 spectrophotometer (Thermo Scientific). On average, cDNA yield was about 4 µg and the specific activity of 30 pmol Cy3 per µg of cDNA.
Microarray experiments were performed using SurePrint G3 Mouse Gene Expression 8x60K microarrays (AgilentTechnologies). 800 ng of labeled cDNA target were mixed with 5 µl of 10X Blocking Agent (AgilentTechnologies) and water to a final volume of 25 µl. Samples were denaturated at 95°C for 2 min. and added to 25 µl of 2X GEx Hybridization Buffer HI-RPM (AgilentTechnologies). 40 µl mix was dispensed onto the array. Slides were loaded into the Agilent SureHyb chambers and hybridization was performed in a hybridization oven at 65°C for 17 hours with 10 rpm rotation. After hybridization, slides were washed using Wash Buffer Kit (Agilent Technologies) and dried at room temperature.
Microarray slides were scanned using G2505C scanner (Agilent Technologies) at 3 µm resolution. Probes features were extracted using the Feature Extraction Software v. 10.7.3.1 with GE_1_Sep09 protocol (Agilent Technologies).
Wild type control matching with 7 days of AAVshluc, AAVshMCU and AAVMCU transfections Fast skeletal muscle fibre, derived from mouse with the same age of the one where MCU was silenced or overexpressed with AAV injection and sacrification after 7 or 14 days after injection, wild type
Intra-array normalizations were directly performed by the Feature Extraction Software. Inter-array normalization of expression levels was performed with quantile method (Bolstad BM et al., 2003) and the values for within-arrays replicate spots were then averaged. Feature Extraction Software, which provided spot quality measures, was used to evaluate the quality and reliability of the hybridization. In particular, the flag “glsFound” (set to 1 if the spot had an intensity value significantly different from the local background and to 0 when otherwise) was used to filter out unreliable probes: the flag equal to 0 was to be noted as “not available (NA).” Probes with a high proportion of NA values were removed from the dataset in order to carry out a more solid and unbiased statistical analyses. Forty-five percent of NA was used as the threshold in the filtering process, and a total of 30,073 of 39,570 probes were obtained. To identify differentially expressed probes in at least one condition one way ANOVA analysis was performed using a threshold p-value ≤ 0.01. Significant diferentially expressed probes were used to search specific expression clusters according to Self Organizing Tree Algorithm (SOTA) (Herrero JA et al., 2001) as implemented in MultiExperiment Viewer version 4.8.1 (tMev) of the TM4 Microarray Software Suite (Saeed AI et al., 2006). Gene ontology analysis was performed using DAVID web tool (Huang DW et al., 2009), while pathway analysis was performed applying Gene Set Enrichment Analysis (GSEA) as implemented in Graphite web tool (Sales G et al., 2013). KEGG pathway database was used and only pathways that presented at least 10 mapped genes in common with our gene expression matrix were considered in the analysis.