|
Status |
Public on May 15, 2007 |
Title |
PANC-1_BiolRep1_TechRep3_2 |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
PANC-1 cell line grown in serum-containing media
|
Organism |
Homo sapiens |
Characteristics |
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
|
|
|
Channel 2 |
Source name |
PANC-1 cell line 24 hours after serum withdrawal
|
Organism |
Homo sapiens |
Characteristics |
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.
|
|
|
Submission date |
May 13, 2007 |
Last update date |
May 14, 2007 |
Contact name |
Margaret C Cam |
E-mail(s) |
maggie.cam@nih.gov
|
Phone |
240-760-7179
|
Organization name |
NIH
|
Street address |
Bldg 37/3041C
|
City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
|
|
Platform ID |
GPL875 |
Series (1) |
GSE7785 |
PANC-1 cell differentiation |
|
Data table header descriptions |
ID_REF |
|
VALUE |
Log2 Ratio of (D13-Cy3)/(C13-Cy5) |
D13-Cy3 |
Day 1 treated, Median normalized, batch removed signal |
Lowess Normalized D13-Cy3 |
Day 1 treated, Lowess normalized |
C13-Cy5 |
Control, Median normalized, batch removed signal |
Lowess Normalized C13-Cy5 |
Control, Lowess normalized |
Ratio |
Ratio of (D13-Cy3)/(C13-Cy5) |