array scanning protocol
Arrays were scanned with a confocal LuxScanscanner, and images were analyzed with SpotData software.
Fluorescent dye-labeled cDNA from each GC sample and common reference were pooled to hybridize with one chip, and hybridization was performed in duplicate with dye reversal approach. Only those spots were accepted for further analysis of which intensity in at least one channel exceed the local background signal plus 3 standard deviations.
Fluorescent dye (cy5 and cy3-dCTP) labeled DNA was produced through a RNA amplification method and subsequent enzymatic reaction.
A total of 158 patients undergoing gastrectomy for potentially curable GC at Wuhan General Hospital of Guangzhou Command were subjects in this study. This study has been approved by the Ethics Committee of Wuhan General Hospital of Guangzhou Command, PLA, and informed consent was obtained from all subjects. Information on clinicopathologic, therapeutic, and outcome parameters of patients from May 2008 to July 2011 was collected retrospectively. None of patients received chemotherapy or radiotherapy before surgery, and eligibility including histologically confirmed adenocarcinoma of the stomach or gastroesophageal junction.
nucleic acid extraction protocol
Total RNA was extracted with TRIZOL reagent (Invitrogen, Gaithersburg, MD) and further purified with a Nucleospin RNA Clean-up Kit (Macherey-Nagel, Germany) according to the protocol supplied by the manuscript.