tissue: stomach cell line: normal gastric fibroblast
Fresh normal gastric tissues were obtained from 2 gastric cancer patients and minced with scalpels in a culture dish. Samples were enzymatically dissociated in 20 ml of D/F12+serum media containing collagenase I in a 37℃ incubation for 12-15 h. After digestion, samples were centrifuged at 700 rpm for 5 min to separate epithelial cells and fibroblast cells. Fibroblast cells (named as NL14 and NL32 from two normal gastric tissues, respectively) were collected from the supernatant by centrifugation at 800 rpm for 8 min, washed twice with PBS, and cultured in D/F12 media supplemented with 10% FBS and 1% antibiotics.
RNA was extracted using RNeasy kit from 2 normal fibroblasts obtained by treatment protocol. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
0.5 ug of total RNA was amplified and labeled using Agilent Quick Amp Labeling Kit. After labeling, cRNA was purified with Qiagen’s RNeasy Mini-spin columns (QIAGEN, Germantown, MD) and quantified using NanoDrop ND-1000 UV-VIS Spectrophotometer for cyanine 3 dye concentration, RNA absorbance ratio (260 nm/280 nm), and cRNA concentration.
Cyanine 3-labled linearly amplified cRNA was mixed with 10X blocking agent, nuclease-free water, and 25X fragmentation buffer provided in the Agilent Gene Expression Hybridization Kit (Agilent Technologies), and incubated for 30 min at 60oC to fragment RNA. After adding 2X GEx hybridization buffer Hi-RPM, the amplified fragmented RNA was hybridized on a Agilent SurePrint G3 Human GE v2 8x60K Microarray Chip using a SureHyb DNA Microarray Hybridization Chamber in a DNA Microarray Hybridization Oven (Agilent Technologies) at 10 rpm, 65℃ for 17 hours.
Slides were scanned with an Agilent DNA microarray scanner (Agilent Microarray Scanner–G2565BA, Agilent Technologies).
Feature Extraction Software v9 (Agilent Technologies) was used to extract and analyze the signals. To validate the amplification process, amplified and unamplified RNAs were labeled, hybridized and compared.