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Status |
Public on Jun 08, 2009 |
Title |
1945002_532 |
Sample type |
RNA |
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Source name |
histologically normal human colon tissue obtained from surgery for adenocarcinoma of the colon
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Organism |
Homo sapiens |
Characteristics |
individual: 1945002 tissue: colon
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Extracted molecule |
total RNA |
Extraction protocol |
From the frozen tissue, 5 micron thick cryosections were cut, mounted on glass slides, and stained with Hematoxylin and Eosin. Sections were reviewed by a pathologist to confirm the diagnoses of prostatic adenocarcinoma and benign tissue. Areas of tumor were selected where >60% of cells consisted of tumor cells. Areas of benign tissue were selected where >50% of cells consisted of non-neoplastic epithelium. From these areas, two 2 mm punch biopsy cores of frozen tissue were removed from the frozen block and processed for DNA and RNA extraction using a modified Qiagen Allprep DNA/RNA protocol. Briefly, tissue was homogenized in Qiagen buffer RLT with b-mercaptoethanol. Lysates were passed through Allprep DNA columns for purification of DNA. All washes and elution of DNA from the Allprep column were performed per manufacturer’s protocol. Initial flow through from the column was mixed with 3 volumes of Trizol LS reagent (Invitrogen) and processed per manufacturer’s protocol to yield total RNA.
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Label |
Cy3
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Label protocol |
RNA was converted to double-stranded cDNA (SuperScript Double Strand cDNA Synthesis Kit, Invitrogen USA, Carlsbad, CA) with oligo-dT primer (Promega USA) according to the manufacturer’s protocol. Resultant ds-cDNA was labeled using cy3-tagged random 9mers and Klenow fragment for 2h at 37°C, prior to denaturation and hybridization to a microarray. Genomic DNA was labeled using cy5-tagged random 9mers.
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Hybridization protocol |
The Cy3- and Cy5-labeled samples were combined (5 ug each) in hybridization buffer, denatured, and hybridized to the custom 8q24 385K feature array using standard hybridization conditions for gene expression (NimbleGen Expression User Guide, Roche NimbleGen, Madison, WI). Slides were then washed and dried using standard conditions per the Roche NimbleGen protocol.
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Scan protocol |
Slides were scanned with a GenePix 4000B Scanner. Initial pre-scannig was performed at PMT Gain = 500 and Power (%) = 100. After the pre-scan, the PMT was adjusted to avoid saturated spots.
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Description |
RNA-normalized signal intensity; a probe was considered positive in the cDNA array when its intensity was higher (> 1) than its genomic DNA probe value.
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Data processing |
Initially, data generated from each group of microarray scans (cDNA and genomic DNA) were normalized separately. Quantile normalization of probe intensities was carried out using the Multi-Array quantile normalization module, part of the NMPP 1.01 package with the two-step normalization option as described by Wang and colleagues (Wang, X. et al. NMPP: a user-customized NimbleGen microarray data processing pipeline. Bioinformatics 2006 22, 2955-7). First, replicate slides within each set were normalized, followed by a global normalization to adjust both sets to an average baseline.
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Submission date |
May 26, 2009 |
Last update date |
Jun 07, 2009 |
Contact name |
Mark Pomerantz |
E-mail(s) |
mark_pomerantz@dfci.harvard.edu
|
Phone |
617-632-1914
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Fax |
617-632-2165
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Organization name |
Dana-Farber Cancer Institute
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Department |
Medical Oncology
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Street address |
44 Binney St.
|
City |
Boston |
State/province |
MA |
ZIP/Postal code |
02115 |
Country |
USA |
|
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Platform ID |
GPL8592 |
Series (1) |
GSE16228 |
RNA expression at chromosome 8q24 in human colon |
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