|
| Status |
Public on Nov 27, 2013 |
| Title |
BT549 vs Cell Line pool |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Breast cancer cell line
|
| Organism |
Homo sapiens |
| Characteristics |
tumor cell line: BT549
tumor type: Invasive ductal carcinoma
tumor source: Primary breast
biomaterial provider: ATCC
pmol cy3/μg crna: 13
μg cy3 probe hybridized: 1.65
|
| Treatment protocol |
Cells were grown to exponential growth phase prior to harvesting.
|
| Growth protocol |
Cell lines from ATCC and Asterand were cultured using conditions recommended by the supplier. TES cell lines were cultured in media optimized by Drs. Felding Habermann and Smider at the Scripps Research Institute, San Diego, CA. Publication pending. Refer to conference proceeding: Cancer Res 2011;71(24 Suppl):Abstract nr PD03-07 for details of cell line development.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell monolayers were washed onle with PBS (calcium and magnesium free) and lysed at room temperature by addition of TRIzol reagent (Invitrogen; Carlsbad, CA). Organic lysate was homogenized by manual pipetting and stored at -80oC. Total RNA was purified from 200 μl TRIzol lysate using the RNeasy micro kit (Qiagen; Valencia, CA) following manufacturer's instructions including the optional DNAse treatment step. Total RNA concentration and purity was measured using a Nanodrop ND-1000 Spectrophotometer (Nanodrop; Wilmington, DE). Average total RNA concentration 770 ng/μl. RNA integrity was evaluated using a 2100 Bioanalyzer (Agilent Technologies; Palo Alto, CA). All samples had an RNA integrity # (RIN#) of 9.4 or higher (median was 9.9).
|
| Label |
Cy3
|
| Label protocol |
Cy3-labeled cRNA probes were generated from 250 ng of RNA from each cell line using Agilent's Low Input Quick Amp Labeling Kit (Agilent Technologies; Palo Alto, CA, Catalog# 5190-2306) following manufacturer's instructions. Probe Incorporation efficiency (pmoL cy dye/μg cRNA) provided. For the reference pool, 200 ng of total RNA from each cell line was combined. 10 batches of Cy5-labeled cRNA reference pool (500 ng total RNA input each) were prepared using Agilent's Low Input Quick Amp Labeling Kit. Each batch with Cy5 incorporation efficiency >8.0 pmoL cy dye/μg cRNA was combined and aliquotted to facilitate controlled batch hybridization. Cy dye incorporation efficiency was calculated with a Nanodrop ND-1000 Spectrophotometer (Nanodrop; Wilmington, DE) following Agilent-recommended protocols. Details for incorporation efficiency (pmoL cy dye/μg cRNA) are provided.
|
| |
|
| Channel 2 |
| Source name |
reference pool
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Pool from all cell lines (reference)
pmol cy5/μg crna: 15.01
μg cy5 probe hybridized: 1.65
|
| Treatment protocol |
Cells were grown to exponential growth phase prior to harvesting.
|
| Growth protocol |
Cell lines from ATCC and Asterand were cultured using conditions recommended by the supplier. TES cell lines were cultured in media optimized by Drs. Felding Habermann and Smider at the Scripps Research Institute, San Diego, CA. Publication pending. Refer to conference proceeding: Cancer Res 2011;71(24 Suppl):Abstract nr PD03-07 for details of cell line development.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell monolayers were washed onle with PBS (calcium and magnesium free) and lysed at room temperature by addition of TRIzol reagent (Invitrogen; Carlsbad, CA). Organic lysate was homogenized by manual pipetting and stored at -80oC. Total RNA was purified from 200 μl TRIzol lysate using the RNeasy micro kit (Qiagen; Valencia, CA) following manufacturer's instructions including the optional DNAse treatment step. Total RNA concentration and purity was measured using a Nanodrop ND-1000 Spectrophotometer (Nanodrop; Wilmington, DE). Average total RNA concentration 770 ng/μl. RNA integrity was evaluated using a 2100 Bioanalyzer (Agilent Technologies; Palo Alto, CA). All samples had an RNA integrity # (RIN#) of 9.4 or higher (median was 9.9).
|
| Label |
Cy5
|
| Label protocol |
Cy3-labeled cRNA probes were generated from 250 ng of RNA from each cell line using Agilent's Low Input Quick Amp Labeling Kit (Agilent Technologies; Palo Alto, CA, Catalog# 5190-2306) following manufacturer's instructions. Probe Incorporation efficiency (pmoL cy dye/μg cRNA) provided. For the reference pool, 200 ng of total RNA from each cell line was combined. 10 batches of Cy5-labeled cRNA reference pool (500 ng total RNA input each) were prepared using Agilent's Low Input Quick Amp Labeling Kit. Each batch with Cy5 incorporation efficiency >8.0 pmoL cy dye/μg cRNA was combined and aliquotted to facilitate controlled batch hybridization. Cy dye incorporation efficiency was calculated with a Nanodrop ND-1000 Spectrophotometer (Nanodrop; Wilmington, DE) following Agilent-recommended protocols. Details for incorporation efficiency (pmoL cy dye/μg cRNA) are provided.
|
| |
|
| |
| Hybridization protocol |
Each Cy3-labeled cRNA sample probe was co-hybridized with Cy5-labeled cRNA reference probe to Agilent 4x44K (V1) expression microarrays following Agilent protocols. Details for μg of cRNA probes co-hybridized are provided.
|
| Scan protocol |
Slides were washed with Agilent's Gene Expression Wash Buffers (Part Number 5188-5327) following Agilent's protocol and scanned at 5 μm using an Agilent Microarray Scanner (model G2505B) in an ozone-controlled environment. Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1). Build hg18.
|
| Data processing |
Array control features and control targets were removed. Data transformed from Log10 to Log2.
|
| |
|
| Submission date |
Sep 23, 2013 |
| Last update date |
Nov 27, 2013 |
| Contact name |
Heather E. Cunliffe |
| Organization name |
University of Otago, Dunedin School of Medicine
|
| Department |
Department of Pathology
|
| Street address |
P O Box 913
|
| City |
Dunedin |
| State/province |
Otago |
| ZIP/Postal code |
9054 |
| Country |
New Zealand |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE51086 |
29 untreated breast cancer cell lines vs. pool of all 29 cell lines. |
|
