|
| Status |
Public on Nov 02, 2011 |
| Title |
S02 Control 14-Af3 |
| Sample type |
RNA |
| |
|
| Source name |
untreated control
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Fisher 344
genotype/variation: Big Blue Transgenic
gender: male
age: 6 weeks
tissue: kidney
|
| Treatment protocol |
Male, 6-week-old Big Blue rats were treated with Aristolochic Acid as its sodium salt at 10.0 mg/kg body weight by gavage (4 ml/kg body weight) 5 times a week for 12 weeks. The animals were sacrificed 1 day after the last treatment. The kidneys were isolated, flash frozen in liquid nitrogen, and stored at - 80°C.
|
| Growth protocol |
All animal procedures followed the recommendations of the NCTR Institutional Animal Care and Use Committee for the handling, maintenance, treatment, and sacrifice of the rats.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNAs were isolated from about 40-50 mg rat kidney tissue suspended in RNA-Later ICE (Ambion Inc., Austin, TX) using mirVanaTM miRNA isolation kit (Ambion). This kit combines the advantages of acid-phenol:chloroform extraction and glass solid-phase extraction and can yield high quality of microRNA-retaining total RNAs. RNA concentrations were determined on NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, Delaware) and the qualities were checked on on an Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA) using an RNA6000 Nano LabChip (Agilent).
|
| Label |
Cy3
|
| Label protocol |
To perform the miRNA array experiment, total RNA (2 to 5 µg) was first size fractionated using a YM-100 Microcon centrifugal filter (Millipore). The small RNAs (< 300 nt) were then 3’-extended with a poly(A) tail using poly(A) polymerase. Thereafter, an oligonucleotide tags was ligated to the poly(A) tail for later staining with fluorescent dye Cy3.
|
| |
|
| Hybridization protocol |
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies).
|
| Scan protocol |
Hybridization images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
CTL_14
|
| Data processing |
Raw intensities experienced the adjustments of data-filtering, log 2 transformation, gene centering and normalization with a cyclic LOWESS (locally weighted regression). MiRNAs with all 6 samples intensities larger than the intensity threshold of 32 were considered detectable.
|
| |
|
| Submission date |
Nov 01, 2011 |
| Last update date |
Nov 02, 2011 |
| Contact name |
Fanxue Meng |
| E-mail(s) |
fanxue.meng@fda.hhs.gov
|
| Phone |
870-543-7082
|
| Organization name |
National Center for Toxicological Research
|
| Street address |
3900 NCTR Road, HFT-130
|
| City |
Jefferson |
| State/province |
AR |
| ZIP/Postal code |
72079 |
| Country |
USA |
| |
|
| Platform ID |
GPL14819 |
| Series (1) |
| GSE33360 |
Discovery of novel microRNAs in rat kidney using microarray analyses |
|
