|
| Status |
Public on May 16, 2016 |
| Title |
Kidney_Homozygous Mutant PUS1_Indiv4 |
| Sample type |
RNA |
| |
|
| Source name |
Kidney, Homozygous Mutant PUS1 Individual 4
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J with backcrosses for 5 generations before experimentation
tissue: Kidney
genotype/variation: Homozygous Mutant PUS1
gender: Male
|
| Treatment protocol |
N/A
|
| Growth protocol |
The tissue samples were from mice that were 14 to 16 weeks old
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from frozen kidney (cross section of whole kidney) using the mirVanaTM PARISTM kit.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) and Cyanine-5 (Cy5) labeled cRNA was prepared from 200 ng RNA using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with a NanoDrop 2000c Spectrophotometer.
|
| |
|
| Hybridization protocol |
300 ng of Cy3-labelled cRNA and 300 ng of Cy5-labelled cRNA was mixed and fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarray (G4852A-028005) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then immersed in acetonitrile and dried immediately by slowly removing the slide.
|
| Scan protocol |
Slides were scanned immediately after washing on an Agilent DNA Microarray Scanner (G2505C) using two color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 um, Dye channels set to 20 bit).
|
| Description |
Gene expression of Kidney tissue
G: US10130359_252800516690_S01_GE2_107_Sep09_2_4.txt
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE2_107_Sep09 and Grid 028005_D_F_20130207) to obtain background- and dye-corrected (LOWESS) signal intensities. Data was uploaded into Genespring version 11.5.1 where it was log2 transformed, quantile normalized and base line transformed using the median of all samples. Data was filted by flags in a way that at least 75% of the samples in any of the experimental groups had DETECTED flags. Then, differentially expressed genes were determined.
|
| |
|
| Submission date |
Feb 11, 2016 |
| Last update date |
May 16, 2016 |
| Contact name |
Diego Altomare |
| Organization name |
University of South Carolina
|
| Department |
Department of Drug Discovery and Biomedical Sciences
|
| Lab |
Functional Genomics Core
|
| Street address |
715 Sumter Street, Room 617
|
| City |
Columbia |
| State/province |
SC |
| ZIP/Postal code |
29208 |
| Country |
USA |
| |
|
| Platform ID |
GPL10787 |
| Series (2) |
| GSE77819 |
Knockout of Pseudouridine synthase 1 (PUS1) in mice. [Kidney] |
| GSE77823 |
Knockout of Pseudouridine synthase 1 (PUS1) in mice. |
|
