Normalization was performed using R. Additional information can be found under http://www.vumc.com/branch/microarrays/5903111/.
Scanning was performed according to the manufacturers instructions (Two-Color Microarray-Based Gene Expression Analysis – Low Input Quick Amp Labeling protocol, version 6.5).
(Parameters: Scanning hardware = OTHER: Agilent C scanner, Scanning software = Scanning software)
Two-Color Microarray-Based Gene Expression Analysis
Low Input Quick Amp Labeling protocol
Version 6.5, May 2010
(Parameters: Amplification = none, Mass unit = Micro gram)
Samples were hybridized onto Agilent Human* GE 4x44K v2 Microarrays (Agilent Technologies) according to the manufacturers instructions (Two-Color Microarray-Based Gene Expression Analysis – Low Input Quick Amp Labeling protocol, version 6.5) not including the RNA Spike-In kit step (Step 1).
(Parameters: Chamber type = OTHER: Agilent SureHyb chamber, Quantity of label target used = 825, Mass unit = Nano gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 65)
This is a pool of the ten carcinoid samples used in the microarray analysis. This pool contains 1/10 of sample 1, 1/10 of sample 2 etc.
RNA was isolated from the cases using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). The quality of the RNA was tested by chip analysis using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).
(Parameters: Extracted product = total_RNA, Amplification = none)
All samples were from resected tumors that were fresh frozen. From these frozen tumors, sections were cut. All sections contained >70% tumor cells, as verified by a pathologist. Total RNA was later isolated using thse sections.
Universal Human Reference RNA (Stratagene, Agilent Technologies, Santa Clara, CA, USA, catalogue number 74000) from a cell line pool expressing 70-80% of all human genes.