|
| Status |
Public on Dec 29, 2016 |
| Title |
1/2_Kidney_7dpi_Replicate1 |
| Sample type |
RNA |
| |
|
| Source name |
1/2 Kidney, 7dpi Replicate 1
|
| Organism |
Mus musculus |
| Characteristics |
tissue: 1/2 Kidney
gender: Female
strain: C3H/HeOuJ
age: 6 weeks
|
| Treatment protocol |
Six week old C3H/HeOuJ (Jackson Laboratories, Bay Harbor, ME) female mice were acclimated on standard low-fat chow (Harlan) with 12 hour light/dark cycles for one week, housed in ventilated cages, transferred to a BL2 room and inoculated transurethrally using 10^8 cfu CFT073. At 0 days (control) and 7 days following infection, steriley dissected longitudinally halved kidneys were snap frozen in liquid nitrogen and stored at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from snap frozen kidneys using the mirVanaTM kit (Life Technologies, Carlsbad, CA) following the manufacturer's recommendations. RNA integrity was analyzed using Agilent 2100 Bioanalyzer Lab-On-A-Chip 6000 Series II chip (Agilent Technologies, Santa Clara, CA)
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared usingAglient’s One-Color Microarray-based Gene Expression Analysis Low Input Quick Amp Labeling according to the manufacturer's instructions, followed by purification using Qiagen's RNAeasy column-based kit. Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5mg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/mg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250mL containing 1X Agilent fragmentation buffer and 2X Agilent blocking according to the manufacturers recommendations. On completion of the fragmentation reaction, 250mL of 2X Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Mouse Gene Expression Microarrays (G4852A) 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 dried immediately by brief centrifugation.
|
| Scan protocol |
Microarray slides were hybridized overnight, washed and then scanned with Agilent G2505C Microarray Scanner. Samples were hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarray and slides were scanned using Agilent G2505C Microarray Scanner.
|
| Description |
Gene expression in 7-days post infection C3H/HeOuJ mouse kidney
|
| Data processing |
The information about each probe on the array was extracted from the image data using Agilent Feature Extraction 10.10 (FE). This data is stored in the FE “.txt” files. The raw intensity values from these files was imported into the mathematical software package “R”, which was used for all data input, diagnostic plots, normalization and quality checking steps of the analysis process using scripts developed in-house by Peter White specifically for this analysis. The dataset was filtered to remove positive control elements and any elements that were flagged as outliers. Present (P), Marginal (M) or Absent (A) calls were made for each element on the array using in-house methodologies that took into account both the results of FE probe detection statistics and the intensities of the negative controls elements on the array. The algorithm for normalization of this Agilent One-Color Analysis consisted of the following: The median green (Cy3) intensities were normalized between the arrays using the Quantile Normalization package in “R” (Bolstad et al., 2003). Quantile normalization is a non-linear probe-level normalization that results in the same empirical distribution of intensities for each array. This is a significantly more robust approach than simply normalizing to the median valueof each array.
|
| |
|
| Submission date |
Jan 04, 2016 |
| Last update date |
Dec 29, 2016 |
| Contact name |
Ashley R Jackson |
| E-mail(s) |
ashley.jackson@nationwidechildrens.org
|
| Phone |
614-355-2879
|
| Organization name |
Nationwide Children's Hospital
|
| Department |
Molecular and Human Genetics
|
| Lab |
Kirk M. McHugh
|
| Street address |
700 Children's Dr
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43205 |
| Country |
USA |
| |
|
| Platform ID |
GPL13912 |
| Series (1) |
| GSE76469 |
Pyelonephritis-Mediated Renal Fibrosis is Associated with Severe Inflammation |
|
