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Status |
Public on Nov 09, 2012 |
Title |
Peripheral Blood, control 1, 14hrs fasting , rep 1 |
Sample type |
RNA |
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Source name |
Peripheral Blood, control, 14hrs fasting , replicate 1
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Organism |
Homo sapiens |
Characteristics |
tissue: whole blood gender: male age (y): 50 disease state: control
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Extracted molecule |
total RNA |
Extraction protocol |
A total of 3ml of peripheral blood was collected in EDTA coated tubes from each participant after overnight fast. Total RNA was extracted from the blood samples using QIAamp RNA Blood mini kit (Qiagen, USA) using the protocol recommended by the manufacturer. For the microarray experiment, the extracted RNA was cleaned up using the RNeasy Mini Elute cleanup kit (Qiagen, USA). RNA was quantified by NanoDrop ND-1000 Spectrophotometer (NanoDrop,Wilmington,DE, USA) , while the quality was determined using a Bioanalyzer 2100 (Agilent Technologies, CA, USA).
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Label |
Cy3
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Label protocol |
cDNA was synthesized from 200ng of total RNA, and cRNA was synthesized from cDNA and incorporated with Cy3-dUTP through in vitro transcription of the cDNA. Labeled probes were purified using RNAeasy mini kit (Qiagen, USA) and Dye incorporation and cRNA yield were verified with the NanoDrop ND-1000 Spectrophotometer and concentrated to the desired volume
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Hybridization protocol |
5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1ul of 25x Agilent fragmentation buffer and 5ul of 10x Agilent blocking agent following the manufacturer’s instructions. 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent G4851A , human genome G3 Hmn GE 8X60K microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
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Scan protocol |
Slides were scanned on the Agilent DNA Microarray Scanner (G2505-64250) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
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Description |
Gene expression in control samples
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Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028004_D_F_20101102) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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Submission date |
Nov 08, 2012 |
Last update date |
Nov 09, 2012 |
Contact name |
prathima arvind |
E-mail(s) |
prathima79@gmail.com
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Fax |
080-27835302
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Organization name |
Thrombosis Research Institute
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Department |
Functional Genomics
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Street address |
#258/A, Bommasandra Industrial Area, Anekal Taluk
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City |
Bangalore |
ZIP/Postal code |
560099 |
Country |
India |
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Platform ID |
GPL13607 |
Series (1) |
GSE42148 |
Global gene expression profile of coronary artery disease in Asian Indians |
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