|
| Status |
Public on May 15, 2007 |
| Title |
PANC-1_BiolRep1_TechRep2_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 (D12-Cy3)/(C12-Cy5) |
| D12-Cy3 |
Day 1 treated, Median normalized, batch removed signal |
| Lowess Normalized D12-Cy3 |
Day 1 treated, Lowess normalized |
| C12-Cy5 |
Control, Median normalized, batch removed signal |
| Lowess Normalized C12-Cy5 |
Control, Lowess normalized |
| Ratio |
Ratio of (D12-Cy3)/(C12-Cy5) |
