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Please use this identifier to cite or link to this item: http://hdl.handle.net/10481/31632

Title: Progressing vs regressing melanoma metastases (GSE24067)
Authors: Carretero Coca, Rafael
Wang, Ena
Engle, Alyson M.
Ascierto, María L.
Liu, Hui
Camacho, Francisco M.
Marincola, Francesco M.
Garrido Torres-Puchol, Federico
Cabrera Castillo, María Teresa
Issue Date: Sep-2010
Abstract: We documented the transcriptional pattern of 3 progressing and 2 regressing synchronous melanoma metastases from the same patient following M-VAX treatment.
Description: [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.
Keywords: Animals
Chordates
Humans
Mammals
Primates
Vertebrates
Dataset
URI: http://hdl.handle.net/10481/31632
Rights : Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License
Appears in Collections:Información complementaria de las Investigaciones (Datos en Bruto)

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GSM592226.xlsMelanoma progressing 12.37 MBMicrosoft ExcelView/Open
GSM592227.xlsMelanoma progressing 22.37 MBMicrosoft ExcelView/Open
GSM592228.xlsMelanoma progressing 32.37 MBMicrosoft ExcelView/Open
GSM592229.xlsMelanoma regressing 12.36 MBMicrosoft ExcelView/Open
GSM592230.xlsMelanoma regressing 22.37 MBMicrosoft ExcelView/Open
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