|
| Status |
Public on Jul 01, 2008 |
| Title |
Hdac3 Floxminus ER Cre+ MEF treated with 1 micromolar tamoxifen for 72hr replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse embryo fibroblast, Hdac3 Fl/-
|
| Organism |
Mus musculus |
| Characteristics |
Background: C57BL/6
Clone: 1G6
Sample source: Mouse embryo fibroblast
|
| Treatment protocol |
MEFs (Hdac3 FL/-ER Cre+) were isolated and treated with 1micromolar 4-hydroxy tamoxifen for 72hr.
|
| Growth protocol |
MEFs were isolated from E13.5-14.5 embryos and cultured in the Dubecco’s modified Eagle’s medium (DMEM) (Cellgro) supplemented with 10% FBS (Geminin Bio-products), 50μg/ml Penicillin-Streptomycin, 2mM L-Glutamine, and 1% non-essential amino acids (Cellgro).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from MEFs that were treated with either AdCre, or with ethanol (solvent for tamoxifen), or with tamoxifen using the Perfect Pure Tissue Kit (5 Prime). All RNA Preps submitted to the VMSR were run on an Agilent 2100 Bioanalyzer to assess RNA integrity. Those samples meeting minimum requirements of RIN value of 7.0 and greater were used to generate targets for hybridization to ABI arrays.
|
| Label |
Digoxigenin
|
| Label protocol |
One (1) ug of Total RNA (30ng mRNA) was used to generate First Strand cDNA using the NanoAmp RT-IVT labeling kit according to manufacturer’s protocol (ABI, Cat#4365715). Following first strand synthesis, second strand synthesis was completed. The resulting cDNA was then purified using an ABI kit provided column and the entire reaction was used in an IVT reaction to generate DIG labeled cRNA. The cRNA was then purified using a kit provided column and assessed for quality on an Agilent Bioanalyzer. All reactions meeting ABI criteria in terms of quantity and size of target produced were fragmented and then used for hybridization.
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| |
|
| Hybridization protocol |
All hybridization reagents, hybridization controls, wash reagents, and chemiluminescent reagents were provided in the ABI CL Detection Kit, part#4342142, and the manufacturer’s protocol was followed. Briefly, the arrays were equilibrated to room temperature and then pre-hybridized with a 1ml volume for 60’ with agitation (100RPM) at 55C per manufacturer’s protocol. During the pre-hybridization, the DIG-labelled targets were fragmented and then stored on ice until the pre-hyb was completed. Once pre-hyb was completed the targets were mixed with the appropriate reagents, including Hybridization controls, and the 0.5ml target was added to the pre-hybridization through the port on the array chamber. The arrays were immediately returned to the 55C Hybridization oven and agitated exactly 16 hours at 100RPM. The arrays were then washed and incubated with Anti-Dig-APAntibody for 20 minutes. Following antibody washes, the arrays were incubated with Chemiluminescence Enhancing Solution, washed a final time and then stored in the last wash buffer at room temperature. Substrate for the chemiluminescence reaction was added to each array individually and then the array was immediately imaged on the 1700 Chemiluminescent Analyzer.
|
| Scan protocol |
Following addition of the chemiluminescence reaction substrate, each array was immediately imaged on the 1700 Chemiluminescent Analyzer. Each imaging was completed in approximately 15 minutes and the images were assessed for QC/QA and a primary analysis was completed by the AB1700 Expression Array System Software (v 1.1.1).
|
| Description |
Hybridization controls were used per Manufacturer’s instruction (see Hybridization procedures).
|
| Data processing |
Data is exported from the 1700 scanner as a tab-delimited file. The raw data includes signal (background-corrected, unnormalized raw signal of the feature), S/N (the signal to noise ratio, expresses the confidence of the feature's etectibility) and Flags (errors in measurement encountered while scanning). A quantile-based normalization of the data was performed with an R script designed by Applied biosystems. This provides the normalization signal intensity (Value data).
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| |
|
| Submission date |
Jan 23, 2008 |
| Last update date |
Apr 28, 2017 |
| Contact name |
Srividya Bhaskara |
| E-mail(s) |
srividya.bhaskara@hci.utah.edu
|
| Phone |
801-213-4219
|
| Organization name |
University of Utah
|
| Department |
University of Utah
|
| Street address |
2000 circle of hope
|
| City |
salt lake city |
| State/province |
Utah |
| ZIP/Postal code |
84112 |
| Country |
USA |
| |
|
| Platform ID |
GPL2995 |
| Series (1) |
| GSE10250 |
Deletion of Histone Deacetylase 3 reveals critical roles in S-phase progression and DNA damage control |
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