|
| Status |
Public on Dec 12, 2009 |
| Title |
BALB MCA+BHT 25 week uninvolved_5315 |
| Sample type |
RNA |
| |
|
| Source name |
mouse RNA from lungs after MCA or MCA + BHT treatment
|
| Organism |
Mus musculus |
| Characteristics |
Male 5-7 wk. old BALB/c (BALB; Tlr4 normal) and C.C3-Tlr4Lps-d/J (BALBLps-d ; Tlr4 dominant negative) mice were purchased from Jackson Laboratories (Bar Harbor, ME).
|
| Biomaterial provider |
Jackson Laboratories
|
| Treatment protocol |
For BHT-induced tumor promotion studies (Protocol 2), 10 ug MCA/gm (Sigma) body wt. was injected ip. on week one, followed by 150 mg BHT/kg or corn oil vehicle control on week two. Five additional weekly 200 mg/kg BHT doses or vehicle control doses were administered to maximize tumor promotion (Brown et al, 1999). The mice were sacrificed 26-28 wks after the MCA injection and thus during the tumor progression stage of carcinogenesis.
|
| Growth protocol |
Mice were allowed to acclimate for 1 wk prior to treatment. All animal use was conducted in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care and approved by the NIEHS Animal Care and Use Committee and the MSU Institutional Animal Care and Use Committee. Mice were housed in shoebox cages in a humidity and temperature-controlled room and provided water and pelleted open-formula rodent diet NIH-07 (Zeigler Brothers, Gardners, PA.) ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the left lung lobe using the RNAeasy Mini Kits (Qiagen, Valencia, CA) and following the kit specifications, including Dnase 1 treatment.
|
| Label |
biotin
|
| Label protocol |
Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3’ Amplification One-Cycle Target labeling kit according to manufacturer’s protocol.
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| |
|
| Hybridization protocol |
15ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol.
|
| Scan protocol |
Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000. Data was obtained using the Genechip® Operating Software (Version 1.2.0.037).
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| Description |
Gene expression analysis was conducted using Mouse 430A Genechip® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3’ Amplification One-Cycle Target labeling kit according to manufacturer’s protocol. For each array, 15ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000. Data was obtained using the Genechip® Operating Software.
|
| Data processing |
Since samples were hybridized to two different microarray platforms (MOE430A or MOE430Av2), data were merged based on common probeset identifiers. Principal component analysis (PCA) was performed to examine the effect of the two platforms. The primary component of variation defined the platform on which a sample was hybridized. This effect appeared independent of the genotype or treatment condition, therefore, singular value decomposition was used to adjust the data and minimize the effect of platform biases (Alter et al. 2000; Nielsen et al. 2002). PCA plots after adjustment gave no evidence of residual platform effect. This adjusted data was further normalized by gc-RMA and used further as described below (Wu et al, 2004).
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| |
|
| Submission date |
Jul 17, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
NIEHS Microarray Core |
| E-mail(s) |
microarray@niehs.nih.gov, liuliw@niehs.nih.gov
|
| Organization name |
NIEHS
|
| Department |
DIR
|
| Lab |
Microarray Core
|
| Street address |
111 T.W. Alexander Drive
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
| GSE8504 |
Transcriptomic analysis of TLR4 pathways in a murine model of chronic pulmonary inflammation and carcinogenesis |
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