|
| Status |
Public on Aug 17, 2020 |
| Title |
Prostate_PND 100 Male_TE_Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Whole Prostate, male, PND 100, T + E, replicate 3
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
tissue: Prostate
gender: Male
age: Postnatal day (PND) 100
treatment: T+E
|
| Treatment protocol |
Sprague-Dawley (Hsd:SD) male rat offspring were administrated estradiol benzoate by subcutaneous injection at PND 1, 3 and 5 and subsequently, treated with additional hormones (estradiol and testosterone) at PNDs 90-145.
|
| Growth protocol |
11–13-week-old time-mated female SD rats were purchased from Envigo (Indianapolis, IN, USA) and were delivered to the National Center for Toxicological Research (NCTR) on GD 3 (day of birth=PND 0). Pregnant dams were housed individually and maintained under a 12:12-h light-dark cycle with controlled room temperature (23°C ± 3°C) and humidity (50% ± 20%). Dams and male pups were fed with low phytoestrogen 5K96 chow (Purina Mills, St. Louis, MO) from gestation day (GD) 3. After birth, male offspring were also kept under same conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA samples from prostate of PNDs 30, 100 and 145 rats (N=3-4 litters/group) were extracted using the miRNeasy Kit (Qiagen, Valencia CA, USA). After extraction, the concentration of each RNA sample was determined using a NanoDrop 2000c spectrophotometer (version 3.0.1, Thermo Fisher Scientific Inc, Wilmington DE, USA). RNA samples with RNA Integrity Numbers (RINs) of 8.0 or above were used for gene expression measurements.
|
| Label |
Cy3
|
| Label protocol |
Total RNAs (500ng) were labeled with cyanine dye (Cy3) using the Agilent One Color Low RNA Input Linear Amplification Kit according to the manufacturer’s protocol (Agilent Technologies, Inc.) followed by RNAeasy column purification (QIAGEN, Valencia, CA). For each reaction, cRNA yields and specific activities were determined using the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
Only the cRNAs with yields >1.65 µg and specific activities > 9.0 pmol of dye per µg cRNA were used for hybridization. Equal amounts (600 ng) of purified Cy3-labeled cRNA were hybridized to Agilent SurePrint G3 rat 8 × 60K microarrays (8 arrays per slide) following the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (V5.5, Agilent Technologies, Inc.) for 17 hours at 65°C in a hybridization oven.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%). The resulting images were analyzed by quantifying the Cy3 fluorescence intensity at each of the 62,976 spots (features) on each array using the Agilent Feature Extraction Software (Version 10.7.3) with default parameters (protocol GE1_107_Sep09) to obtain background subtracted and spatially detrended Processed Signal intensities. The median fluorescence intensity of all the pixels within each feature was taken as the intensity value for that feature.
|
| Description |
Total RNA from Prostate of PND 100 male rats exposed postnatally to T+E was used for gene expression
|
| Data processing |
A total of 33 raw data files for 33 samples (3-4 arrays for each control and treated rats) were obtained from the Agilent Feature Extraction Software. An excel spreadsheet containing raw intensity data for all 33 samples was used as an input file for further data preprocessing and normalization using SAS 9.4 (SAS institute Inc., Cary, NC). There were 62,976 features (probes) on each array. Controls were removed and then data was normalized using 75th percentile scaling.
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| |
|
| Submission date |
Feb 25, 2020 |
| Last update date |
Aug 18, 2020 |
| Contact name |
Vikrant Vijay |
| E-mail(s) |
[email protected]
|
| Phone |
870-543-7525
|
| Organization name |
US FDA National Center for Toxicological Research
|
| Department |
Division of Systems Biology
|
| Lab |
Personalized Medicine
|
| Street address |
3900 NCTR Road
|
| City |
Jefferson |
| State/province |
AR |
| ZIP/Postal code |
72079 |
| Country |
USA |
| |
|
| Platform ID |
GPL22740 |
| Series (1) |
| GSE145917 |
Gene expression profiling in dorsolateral prostates of prepubertal and adult Sprague-Dawley rats dosed with Estradiol Benzoate, Estradiol and Testosterone |
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