Resultados de investigación

Synthetic and crystallographic details, CShMs, and various structural figures and tables., Peer reviewed

Experimental and theoretical details and 1H NMR and 13C NMR spectra., Peer reviewed

The dataset contains detailed information on adolescents (ages 11–16) of both sexes. Each record includes demographic, anthropometric, biochemical, and metabolic variables. Key categories include Sex (Female/Male) and Age, body measurements such as weight, height, body fat percentage, and BMI, biochemical parameters (fasting glucose, triglycerides, total cholesterol, LDL, HDL, HbA1c, fasting insulin), waist and hip circumferences and their ratio, blood pressure (systolic and diastolic), and several indices related to insulin resistance and sensitivity (HOMA-IR, McAuley, Quicki, G_I_ratio, Inv_HOMAIR, SPISE). The dataset also includes derived ratios and metrics that help assess body composition and insulin sensitivity. Overall, it provides a comprehensive view of the nutritional and metabolic status of adolescents, useful for studies on growth, obesity, and cardiometabolic risk., Se financió una primera parte con fondos de un proyecto del plan nacional "Spanish Interministerial Commission of Science and Technology (CYCIT, AGL2011-23810)" y una segunda con un proyecto de la UGR "Andalusia 2014–2020 European Regional Development Fund (ERDF) Operative Program, grant number B-AGR-287-UGR18"., Peer reviewed

Figure_S1: Methods accuracy for the Test Set. The X-axis represents the accumulative number of ‘n’ first predicted interactions, sorted by the corresponding method score, which is different in each case. The Y-axis represents the accuracy, calculated as the ratio of true positives (TP) and total number of predictions considered in the test set extracted from our gold standard of functional associations (see Methods). I2H stands for in silico two-hybrid, MT for mirrortree, PP for phylogenetic profiles, GC stands for gene context, GF stands for gene fusion and AODE for Averaged One Dependence Estimators. -- Table S1: Performance of different classifiers for the Test Set. This table shows performance related descriptors for a number of different classifiers. The descriptors included are: Area Under the ROC Curve (AUC), Mathews Correlation Coefficient (MCC, formula shown below), True Positives (TP), True Negatives (TN), False Positives (FP) and False Negatives (FN). The table is divided in three regimes. The first one (yellow background) represents the incremental inclusion of features in AODE classifiers. New features are included from the most to the least discriminative (MCC score for these features) ones: Methods (Gene Fusion, Gene Context, Phylogenetic Profiles, Mirror Tree and in silico two-hybrid), Length (protein sequence lengths) and Nseqs (number of sequences). Rankings in the list of scores for each method are finally included (as they are derived from the corresponding methods) to build the presented APPIA classifier. The second regime (white background) shows the performance for the AODE using all the features and with flags instead of missing values. Finally, the third regime (green background) shows the performance of the other seven different classifying algorithms used in the preliminary test. MCC = (TP×TN−FP×FN)/SQRT((TP + FN)×(TP + FP)×(TN + FP)×(TN + FN)). -- Table S2: Attributes statistics. This table shows some statistical measures of each attribute used in the classification process. The range of the column values represents the minimum and maximum value reached for this attribute in all the examples. In each case, the mean and the standard deviation are calculated without taking into account the instances with an unknown value. Total number of instances: 2,665,180. It should be noted that the high percentage of unknown values is important in many attributes. -- Table S3: List of fully sequenced genomes used. This is the set of 118 prokaryotic genomes to which the computational prediction method has been applied. -- Text S1: AODE detailed description. A detailed description of the AODE algorithm., [Background] Molecular biology is currently facing the challenging task of functionally characterizing the proteome. The large number of possible protein-protein interactions and complexes, the variety of environmental conditions and cellular states in which these interactions can be reorganized, and the multiple ways in which a protein can influence the function of others, requires the development of experimental and computational approaches to analyze and predict functional associations between proteins as part of their activity in the interactome., [Methodology/Principal Findings] We have studied the possibility of constructing a classifier in order to combine the output of the several protein interaction prediction methods. The AODE (Averaged One-Dependence Estimators) machine learning algorithm is a suitable choice in this case and it provides better results than the individual prediction methods, and it has better performances than other tested alternative methods in this experimental set up. To illustrate the potential use of this new AODE-based Predictor of Protein InterActions (APPIA), when analyzing high-throughput experimental data, we show how it helps to filter the results of published High-Throughput proteomic studies, ranking in a significant way functionally related pairs. Availability: All the predictions of the individual methods and of the combined APPIA predictor, together with the used datasets of functional associations are available at http://ecid.bioinfo.cnio.es/., [Conclusions] We propose a strategy that integrates the main current computational techniques used to predict functional associations into a unified classifier system, specifically focusing on the evaluation of poorly characterized protein pairs. We selected the AODE classifier as the appropriate tool to perform this task. AODE is particularly useful to extract valuable information from large unbalanced and heterogeneous data sets. The combination of the information provided by five prediction interaction prediction methods with some simple sequence features in APPIA is useful in establishing reliability values and helpful to prioritize functional interactions that can be further experimentally characterized., Peer reviewed

The item is made of 2 files, of which 1 is the dataset and the other include detailed information about measures realized.-- Dataset contributed to the Project Carbochange, This dataset gathers discrete measurements of CO2 (pH and alkalinity) and hydrographic variables (salinity, temperature, dissolved oxygen, nitrate, phosphate and silicate) obtained during the cruise MOC2EQ carried out from 5 April to 16 May 2010. pH was measured spectrophotometrically following the Clayton and Byrne (1993). This method consists on adding a dye solution to the seawater sample, so that the ratio between two absorbances at two different wavelengths is proportional to the sample pH. Alkalinity was measured using an automatic potentiometric titrator Titrando 809 Metrohm, with a Metrohm 6.0232.100 combination glass electrode and a Pt-1000 probe for temperature measurement following the methodology given by Pérez and Fraga (1987). Dissolved oxygen was analyzed following the widely applied Winkler method. Determinations of nitrate, phosphate and silicate were carried out following methods described by Hansen and Grassoff (1983) Salinity and Temperature were recorded with a CTD probe, Spanish Projects GHG4MOC (CTM2009-07574-E) and MOC2 (CTM2008-06438-C02-01/MAR) supported by Ministerio de Ciencia e Innovación, and CARBOCHANGE (264879), Clivar_A06_2010.exc.csv.-- Report_MOC2EC_2010.pdf.-- Readme_Clivar_A06_2010.txt, Peer reviewed

The item is made of 2 files, of which 1 is the dataset and the other include a small description of the measured variables.-- Dataset contributed to the Project Carbochange, This dataset gathers discrete measurements of CO2 (pH and alkalinity) and hydrographic variables (salinity, temperature, dissolved oxygen, nitrate, phosphate and silicate) obtained during the cruise MOC2Austral carried out from 8 February to 10 March 2010. pH was measured spectrophotometrically following the Clayton and Byrne (1993). This method consists on adding a dye solution to the seawater sample, so that the ratio between two absorbances at two different wavelengths is proportional to the sample pH. Alkalinity was measured using an automatic potentiometric titrator Titrando 809 Metrohm, with a Metrohm 6.0232.100 combination glass electrode and a Pt-1000 probe for temperature measurement following the methodology given by Pérez and Fraga (1987). Dissolved oxygen was analyzed following the widely applied Winkler method. Determinations of nitrate, phosphate and silicate were carried out following methods described by Hansen and Grassoff (1983) Salinity and Temperature were recorded with a CTD probe, Spanish Projects GHG4MOC (CTM2009-07574-E) and MOC2 (CTM2008-06438-C02-02/MAR) supported by Ministerio de Ciencia e Innovación, and CARBOCHANGE (264879), Clivar_A21_2010.exc.csv.-- Metadata_MOC2Austral_2010.xls.-- Readme_Clivar_A21_2010.txt, Peer reviewed

The item is made of 2 files, of which 1 is the dataset and the other include a small description of the measured variables.-- Dataset contributed to the Project Carbochange.-- More information in http://hdl.handle.net/10261/115861, This dataset gathers discrete measurements of CO2 (pH and alkalinity) and hydrographic variables (salinity, temperature, dissolved oxygen, nitrate, phosphate and silicate) obtained during the cruise OVIDE 2010 carried out from 8 to 30 June 2010. pH was measured spectrophotometrically following the Clayton and Byrne (1993). This method consists on adding a dye solution to the seawater sample, so that the ratio between two absorbances at two different wavelengths is proportional to the sample pH. Alkalinity was measured using an automatic potentiometric titrator Titrando 809 Metrohm, with a Metrohm 6.0232.100 combination glass electrode and a Pt-1000 probe for temperature measurement following the methodology given by Pérez and Fraga (1987). Dissolved oxygen was analyzed following the widely applied Winkler method. Determinations of nitrate, phosphate and silicate were carried out following methods described by Hansen and Grassoff (1983) Salinity and Temperature were recorded with a CTD probe, French OVIDE Project supported by CNRS, and CARBOCHANGE, Peer reviewed

Base de datos Phinachi. Estructura y datos de la base creada para el proyecto PHINACHI, Fuentes documentales para el estudio del Patrimonio Histórico Inmueble de Navalcarnero y Chinchón, CAM-Ref. 1659/09.

[Organism] Homo sapiens, [Experiment type] Expression profiling by array, [Overall design] Two-condition experiment, Progressing vs regressing metastases. 3 progressing and 2 regressing metastases extracted from the same patient after M-VAX immunotherapy. One replicate per array., [Platforms (1)] GPL7088 CCDTM Hs_CCDTM36k - version 1, [Relations] BioProject PRJNA130231, 1. Sample GSM592226. Title: melanoma progressing 1. Sample type: RNA. Platform ID: GPL7088. Series: GSE24067 Progressing vs regressing melanoma metastases; GSE26383 Response to immunotherapy in melanoma metastasis., Channel 1: [Source name] melanoma, progressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., Channel 2: [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., [Hybridization protocol] 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis non responding after M-VAX immunotherapy 1 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction., 2. Sample GSM592227. Title: melanoma progressing 2. Sample type: RNA. Platform ID: GPL7088. Series (2): GSE24067 Progressing vs regressing melanoma metastases. GSE26383 Response to immunotherapy in melanoma metastasis., Channel 1 [Source name] melanoma, progressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., Channel 2 [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., [Hybridization protocol] 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis non responding after M-VAX immunotherapy 2 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction., 3. Sample GSM592228. Title: melanoma progressing 3. Sample type: RNA. Platform ID: GPL7088. Series (2): GSE24067 Progressing vs regressing melanoma metastases; GSE26383 Response to immunotherapy in melanoma metastasis, Channel 1 [Source name] melanoma, progressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., Channel 2 [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., [Hybridization protocol] 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis non responding after M-VAX immunotherapy 3 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction., 4. Sample GSM592229. Title: melanoma regressing 1. Sample type: RNA. Platform ID: GPL7088. Series (2): GSE24067 Progressing vs regressing melanoma metastases; GSE26383 Response to immunotherapy in melanoma metastasis., Channel 1 [Source name] melanoma, regressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., Channel 2 [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., Hybridization protocol 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis responding after M-VAX immunotherapy 1 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction., 5. Sample GSM592230. Title: melanoma regressing 2. Sample type: RNA. Platform ID: GPL7088. Series (2): GSE24067 Progressing vs regressing melanoma metastases; GSE26383 Response to immunotherapy in melanoma metastasis., Channel 1 [Source name] melanoma, regressing [Organism] Homo sapiens [Characteristics] tissue: melanoma treatment protocol: M-VAX preservation: cryopreservation patient: 1 [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy5 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., Channel 2 [Source name] Total RNA from pooled PBMC [Organism] Homo sapiens [Characteristics] tissue: Blood preservation: cryopreservation reference: Total RNA from pooled PBMC [Extracted molecule] total RNA [Extraction protocol] Total RNA extracted using Qiagen's RNEasy Mini kit following manufacturer's instructions [Label] Cy3 [Label protocol] 0.5 µg of total RNA was primed with 0.25 ug oligo dT-T7 primer (5’ AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3’) at 70°C for 3 min, then reverse transcribed at 42°C for 90 min in the presence of 200 U SuperScript II RTase (Invitrogen), and 1 mM dNTP. Second strand synthesis was done with the 5’ primer switch method using 0.25 ug Ts/SMARTII primers (5’ AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3’) for priming at 65°C for 1 min, and subsequent primer extension at 75°C for 30 min using the Advantage cDNA Polymerase Mix (Clontech) and 0.2mM dNTP. Double stranded cDNA subjected to in vitro transcription at 42°C for 6h using Megascript T7 kit (Ambion). After two rounds of linear amplification, cRNA was chemically labeled using ULS Cy3/Cy5 Fluorescent Labeling Kit (Kreatech), and fragmented by Fragmentation Reagents kit (Ambion)., [Hybridization protocol] 6 ug of labeled, fragmented cRNA per sample was applied to microarrays in Kreatech's Hybridization buffer, and hybridized for 16 h at 42C. After hybridization, slides were washed three four times with wash solutions containg gradually decreased concentrations of SSC (1x-0x) [Scan protocol] Scanned on an Agilent DNA Microarray Scanner. Images were quantified using Agilent Feature Extraction Software (version A.9.5.3). [Description] metastasis responding after M-VAX immunotherapy 2 [Data processing] LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used for feature extraction., We documented the transcriptional pattern of 3 progressing and 2 regressing synchronous melanoma metastases from the same patient following M-VAX treatment.