Proteome and Transcriptome in Irradiated Glands and were monitored continuously for any signs of distress. Animals were kept at 2262uC. Washington and Finnigan, San Jose, CA, USA) and Pep-Miner [18] searching against the rat part of the Uniprot database. Irradiation treatment Salivary glands were locally irradiated with a single dose of 15Gy of X-rays delivered by Clinac DBX linear accelerator with 6 MV photon energy (Varian Medical Systems, Palo Alto, CA, USA). This dose is known to induce sufficient functional damage without compromising the general health of the animals. Rats were anesthetized with ketamine (100 mg/ml) and xylazine (20 mg/ml) at 100 ml/100 g body weight. Rats were placed on their backs facing up and the IR field was determined such that only the head and neck regions were exposed. Control animals were anesthetized and placed in a box but were not irradiated. Saliva collection and preparation Whole saliva was collected from anesthetized animals before and after irradiation (4–9 animals per group) for 15 min after subcutaneous administration of pilocarpine (2 mg/kg body weight). Saliva samples were immediately put on ice and thereafter were centrifuged at 14,000 g for 20 min at 4uC to remove insoluble materials, cell debris and food remnants. Saliva quantity was determined gravimetrically, assuming a density of 1 g/ml. Whole saliva secretion was measured before and 4, 8 and 12 weeks post-IR. Saliva flow rate was normalized to body weight. Saliva control values were set at 100%, and saliva outputs of IR animals were presented as percentage of the mean control value. Protein concentration was determined using the Bio-Rad protein assay according to Bradford [16] (Bio-Rad, Hercules, CA, USA). 2-DE (two-dimensional gel electrophoresis) analysis Saliva samples pooled from at least four animals per time point, repeated at least twice, were frozen at 280uC and lyophilized overnight. Sediments were dissolved in 7 M urea, 2 M thiourea and 4% (w/v) CHAPS and stored at 280uC until analysis. For analytical gels, samples of 45 mg protein were rehydrated and subjected to isoelectrofocusing on 18-cm long pH 3–10 NL Immobiline DryStrips gels (Amersham Biosciences, Uppsala, Sweden) as we previously described [17]. 2-DE Imaging Gels were scanned using a computerized GS-800 calibrated densitometer (Bio-Rad) and protein spots were detected and quantified using Redfin software (Ludesi, Malmo, Sweden). Several limitations exist in 2D gel analysis as a result of gel-to- gel variation as well as variability in the staining method. To overcome these limitations, all gels were subjected to the same conditions simultaneously for the first and second dimensions. Normalization was performed by the total density in image method of Redfin to quantify spot intensities and minimize staining variation between gels. Mass spectrometry (MS) identification and database search For MS identification, a 2-DE gel containing 500-mg protein samples was fixed in 50% (v/v) ethanol, 12% (v/v) acetic acid for 2 h. Proteins were visualized by staining for 16 h with Coomassie Brilliant Blue G250 (Fluka, Buchs, Switzerland), followed by destaining in 20% ethanol. Electrophoretically separated protein spots were selected by matching to those on a silver-stained 2-D gel and were excised under sterile conditions, as previously described [17]. The MS data were clustered and analyzed using Sequest software (version 3.31; J. Eng and J. Yates, University of RNA isolation Before IR and 4, 8 and 12 weeks post-IR, rats were sacrificed and SSGs were removed for RNA isolation using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufac- turer’s instructions. RNA concentration was measured using a Nanodrop ND-100 spectrophometer. Microarray Total RNA (1 mg) from samples pooled from at least four animals per time point was amplified and labeled with a fluorescent dye (either Cy3 or Cy5) using the Low RNA Input Linear Amplification & Labeling kit (Agilent Technologies, Palo Alto, CA, USA) following the manufacturer’s protocol. The amount and quality of the resulting labeled cRNA was measured in a Nanodrop ND-100 spectrophometer. Equal amounts of Cy3- or Cy5-labeled cRNA were hybridized to the 4644 K Agilent Whole Rat Genome Oligo Microarray for 17 h at 60uC in an Agilent DNA-Microarray Hybridization Oven. The arrays were then washed using the Gene Expression Wash Buffer Kit from Agilent. The microarray was scanned using a GenePix4000B Scanner (Axon, Union City, CA, USA) and the data were extracted from the resulting images using GenePix Pro 4.1 software (Axon). The result files (gpr) produced by GenePix were analyzed utilizing the LIMMA [19] software package, available from CRAN site (http://www.r-project.org). Spots flagged as not found or absent in GenePix were removed by filtering. The Cy5 and Cy3 intensities within each array were normalized using the print-tip locally weighted scatter plot smoothing (LOESS) function, with no background correction applied. To identify differentially expressed genes, a parametric empirical Bayesian approach implemented in LIMMA was used [19]. A moderated t test was performed in parallel, with the use of a false discovery rate [20] correction for multiple testing. LIMMA calculated an emission intensity A value [A = (log2(Cy5)6Cy3)/2] where Cy5 and Cy3 are the normalized emission intensities of each feature. A confidence level of P,0.05 was used to pinpoint significantly differentiated genes. Genes had to have an A value (average expression level for the gene across all arrays and channels) of more than 8.5, thus excluding faint emissions. The differentially expressed genes for each binary comparison retrieved by LIMMA were clustered using EXPANDER software [21]. A functional analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and functional categories was performed for all genes using David online software [22]. Real-time PCR Total RNA from at least three animals per time point was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied BioSystems/Ambion, Austin, TX, USA) according to the manufacturer’s instructions. cDNA was amplified using the 7300 real-time PCR system (Applied Biosystems) with TaqMan gene expression assay for a-amylase and cyclin D1 genes, Cdh22, p21, p57, CysS, Psp, Prb1, Muc19, Mgmt, Ywhaq, and c-KIT and GAPDH genes (Applied Biosystems). Data were analyzed with 7300 System SDS Relative Quantifica- tion Software version 1.4.0.25 (Applied BioSystems). PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40636