Human pancreatic carcinoma, epithelial-like cell line Gender: Male
Biomaterial provider
ATCC
Treatment protocol
To induce ICA formation, Serum-containing medium (SCM) was removed, cells were exposed for 60–120 s to 0.05% trypsin (CellgroMediatech) at 25°C, to loosen but not detach the cells from their extracellular matrix (ECM) and then cultured in Serum Free Medium (SFM). SFM was DME/F12 medium containing 17.5 mM glucose, 1% BSA (no. 152401, ICN) and insulin— transferrin—selenium (GIBCO).
Growth protocol
PANC-1 cells (American Type Culture Collection, Manassas, VA) were started as passage 1 (passages 3–6 were used for all experiments). Cells were cultured in serum-containing media (SCM), a mixture of DMEM (Cellgro, Mediatech, Herndon, VA) containing 10% FBS.
Extracted molecule
total RNA
Extraction protocol
TRIzol to isolate total RNA, followed by cleanup using QIAGEN RNeasy Mini Kit.
Label
Cy5
Label protocol
10 ug of RNA was labeled using the Agilent Fluorescent Direct Label Kit
Human pancreatic carcinoma, epithelial-like cell line Gender: Male
Biomaterial provider
ATCC
Treatment protocol
To induce ICA formation, Serum-containing medium (SCM) was removed, cells were exposed for 60–120 s to 0.05% trypsin (CellgroMediatech) at 25°C, to loosen but not detach the cells from their extracellular matrix (ECM) and then cultured in Serum Free Medium (SFM). SFM was DME/F12 medium containing 17.5 mM glucose, 1% BSA (no. 152401, ICN) and insulin— transferrin—selenium (GIBCO).
Growth protocol
PANC-1 cells (American Type Culture Collection, Manassas, VA) were started as passage 1 (passages 3–6 were used for all experiments). Cells were cultured in serum-containing media (SCM), a mixture of DMEM (Cellgro, Mediatech, Herndon, VA) containing 10% FBS.
Extracted molecule
total RNA
Extraction protocol
TRIzol to isolate total RNA, followed by cleanup using QIAGEN RNeasy Mini Kit.
Label
Cy3
Label protocol
10 ug of RNA was labeled using the Agilent Fluorescent Direct Label Kit
Hybridization protocol
Each Agilent slide contained two sets of coordinate arrays, where samples labeled with the opposite dye (Cy5 and Cy3) configurations were hybridized using the Agilent cDNA Microarray Kit Hybridization Protocol
Scan protocol
Slides were scanned using the Agilent G2566AA scanner
Description
RNA extracted from the first biological replicate was derived from 3 culture dishes producing enough sample for three technical replicates, whereas RNA from the second and third biological replicates were derived from one culture dish each.
Data processing
For the Agilent cDNA arrays, the default settings of the Agilent G2566AA Feature Extraction Software (v.A.5.1.1) were used, which selects the LOWESS (locally weighted linear regression curve fit) normalization method. A second round of normalization included median scaling followed by batch (including dye, RNA extraction batch, slide configuration and array) effect removal using Partek Genomics Solution software.