by the Institutional Committee for Use and Care of Laboratory Animals at the University of Miami. RNA Isolation for Gene Arrays To isolate total RNA from the each of the 40 iliac artery samples, 0.3 mL of TRI Reagent (Molecular Research Center Cat #TRI-118) was added per tissue sample. The sample was then macerated using a Kinematica AG Polytron PT-2100 for 1 min. After a 5 min incubation at room temperature 0.2 volumes of chloroform was added and mixed, following by a 3 min incubation at room temperature and 10 min centrifugation at 12,000 g 4uC. The top aqueous layer was transferred to a new tube containing 0.5 volume of isopropanol and 2 to 10 ul of polyacryl carrier (Molecular Research Center, Cat # PC152). After mixing, the precipitate containing the total RNA was collected by 15 min of centrifugation at 12,000 g 4uC. The pellet was then washed two times with 70% ethanol. Total RNA was additionally purified with RNeasy Mini Kit (Qiagen, Cat # 74106). Total RNA yield was determined spectrophotometrically. RNA Qualification by Agilent To characterize the quality of an RNA sample, it was analyzed on the Agilent 2100 Bioanalyzer. Briefly, approximately 100 to 200 ng of the sample was placed in an RNA LabChip with the appropriate sample loading buffer and electrophoresed in parallel with an RNA standard ladder (Ambion cat # 7152). Agilent Technologies’ Bioanalyzer software was used to analyze the results and to create an electropherogram. RNA was considered usable if ratio of 28S ribosomal peak to 18 S ribosomal peak was above 1.0. All the 40 RNA samples used in our expression study had RIN values between 6.4 and 7.2. Labeling and Hybridization 10 to 20 ug of total RNA were mixed with the 200 pMol of oligo-dT primer. After 10 min incubation at 70uC and 5 min at 4uC, 20 units of Transcriptor reverse transcriptase (RuC he, Cat # 3531287001), 10 nMol of each dNTP and 4 nMol of aminoallyl- dUTP (Ambion, Cat # 8439) were added, and this mix was incubated 2 hours at 42uC. After 30 min RNase treatment cDNA was purified using QIAquick PCR purification Kit (Qiagen, Cat # 28106). Amount of cDNA was determined spectrophotometrically, and samples were dried on speedvac. cDNA was resuspended in the carbonate buffer (pH 9.029.3) and mixed with the Cy3- and Cy5-NHS ethers (Amersham, Cat # PA23001 and PA25001, respectively). After 1 hour incubation in the dark 20 mMol of hydroxylamine were added to quench the reaction, and labeled cDNA was purified using QIAquick PCR purification Kit. Concentration of the labeled cDNA and labeling efficiency were determined spectrophotometrically, and labeled cDNA were hybridized to Agilent Whole Rat Genome Arrayfor 17 hours at 60uC according to manufacturer’s instructions. Two samples were hybridized per chip. Therefore, the 40 samples were run on 20 arrays. Image Analysis and Data Processing The microarrays were scanned at 5 micron resolution using a GenePix4000B scanner (Axon Instruments at Molecular Devices) and the resulting images were analyzed with the software package GenePixPro 6.0 (Axon Instruments at Molecular Devices). Data extracted from the images were transferred to the software package Acuity 4.0 (AxonInstruments) for normalization and statistical analysis. Each array was normalized for signal intensities across the whole array and locally, using Lowess normalization. Vascular Myl9 with Injury and Aging Features for further analysis were selected according to the following quality criteria: (1) at least 90% of the pixels in the spot had intensity higher than background plus two standard deviations; (2), there were less than 2% saturated pixels in the spot; (3) signal to noise ratio (defined as ratio of the background subtracted mean pixel intensity to standard deviation of back- ground) was 3 or above for each channel; (4) the spot diameter was between 110 and 150 micron; (5)the regression coefficient of ratios of pixel intensity was 0.6 or above. All data is MIAME compliant and all raw .gpr files from the 20 arrays are deposited in NCBI’s GEO database (GSE29255), a MIAMI compliant database. Gene Arrays: Global Transcription Analysis All 20 raw gpr files (corresponding to the 40 samples of iliac arteries) were imported into GeneSpring GX 11 software (Silicon Genetics, Redwood City, CA). Each condition had 324 replicates/arrays. Normalized expression values were calculated by the Robust Multi-array Average (RMA) method. The resultant signal information was analyzed using one-way analysis of variance (ANOVA) (p,0.05), assuming normality and equal variances. No correction for multiple comparisons was done. GeneSpring’s cross gene error model, which determines the likelihood of observing a specific fold change to the likelihood of observing a fold measurement by the 50.0 th percentile of all measurements in that sample, then setting the average value of expression level for each gene across the samples to 1.0, and plotting the resulting normalized signal value for each sample (values below 0.01 were set to 0.01). The list and the order of various genes in which they appear in the heatmaps can be viewed in tabular form in the supplemental files. Analysis of Previous Aging Aortas Microarray Datasets Similarly, we performed extensive analysis on a publicly available microarray experiments on aging rat thoracic aortas: (A) Sixteen chips from the rat thoracic aorta tissue from 3, 6, 18, and 28 month old rats (n = 4 per group) [8] (GEO Accession: GSE7281). All 16 raw expression files were normalized using RMA processor. All normalized expression data was analyzed using GeneSpring software. Following normalization one-way analysis of variance was performed for each gene to identify statistically significant gene expression changes. Two criteria were used to determine whether a gene was differentially expressed: fold change of 62.0 and p value ,.05 using a two-tailed distribution. No correction for multiple comparisons was done. Lists of differentially expressed genes from different experiments were compared within GeneSpring and displayed as Venn diagrams to show overlapping and non-overlapping genes (Figure 3). As in our previous work [29], the convergence of differentially expressed genes (Venn diagrams) is what was used to replace the multiple testing in further screening the genes that passed the ,.05 significance levels. Immunohistochemistry Cross sections were taken from different levels of the paraffin- embedded arteries. After tissue rehydration, endogenous peroxi- dase was blocked with 3% hydrogen peroxide. Epitope retrieval was performed with citrate buffer (10 mM sodium citrate, pH 6.0) in a Pascal chamber. Non-specific binding was blocked with 0.5% blocking solution (DAKO, Carpinteria, CA). Slides with the Myl9 primary antibody (1:250, Abcamab64161)were incubated for 1 hour at room temperature (RT). Biotinylated secondary antibodies (DAKO Universal link) were applied for 30 minutes, followed by a washing step with PBS and 15 min incubation with PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e25855