Total RNA was extracted each from 300uL serum samples using 3D-Gene® RNA extraction reagent from liquid sample kit (Toray Industries, Inc.).
Label
Cy5
Label protocol
miRNA was labeled using 3D-Gene® miRNA Labeling kit in accordance with the manufacturer's instructions.
Hybridization protocol
Hybridized for 16 h at 32 ºC with rotary shaker (250 rpm). Hybridization buffer and washing protocol was attached in the 3D-Gene® miRNA oligo chip kit.
Scan protocol
3D-Gene® Scanner (Toray Industries, Inc.) was used for scanning. Images were quantified using Extraction version 2.0.0.6 (Toray Industries, Inc.).
Data processing
The presence of miRNA was determined based on a corresponding microarray signal of greater than [the mean + 2 × standard deviation] of the negative controls signal, of which the top and bottom ranked ones by signal intensity were removed. Once a miRNA was considered present, the mean signal of the negative controls of which the top and bottom 5% ranked by signal intensity were removed was subtracted from the miRNA signal. When the signal value was negative (or undetEsophageal Cancerted) after background subtraction, the value was replaced by 0.1 on a base 2 logarithm scale.
