|
| Status |
Public on Oct 01, 2010 |
| Title |
Kidney Mouse05_Sample01 |
| Sample type |
RNA |
| |
|
| Source name |
Kidney Mouse05_Sample01
|
| Organism |
Mus musculus |
| Characteristics |
tissue type: left kidney
strain: C57BL/6J
gender: male
body weight (g): 22.5
age (days): 69
|
| Treatment protocol |
At 10 weeks, body weight was recorded and the mice were euthanized by cervical dislocation. Dissection procedures were started at 11:00 a.m. after a 4-hour fasting period and were completed within a one-hour time window.
|
| Growth protocol |
We obtained twelve C57BL/6J male mice from The Jackson Laboratory. Six pairs of littermates were pair-housed from weaning and put on a 5k52 diet (standard chow containing 6% fat).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Inguinal fat pad, heart, liver, and both kidneys were extracted, perfused with RNase-free DEPC-treated PBS, cut into pieces not exceeding 0.5 cm in any dimension, and placed in 15 ml conical tubes. The pieces were divided into 2 aliquots and stored in RNAlater (Ambion, Austin TX). Each kidney aliquot consisted of one complete kidney. Tissues were homogenized in TRIzolTM (Invitrogen, Carlsbad, CA). Total RNA was isolated by standard TRIzolTM methods according to the manufacturer’s protocols, and quality was assessed using an Agilent 2100 Bioanalyzer instrument and RNA 6000 Nano LabChip assay (Agilent Technologies, Santa Clara, CA). The RNA was then treated with DNase1 (Qiagen, Valencia, Ca.) according to the manufacturer’s methods. Total RNA was then reverse transcribed followed by second strand cDNA synthesis.
|
| Label |
biotin
|
| Label protocol |
Illumina Sentrix ® Mouse-6 v1.1 BeadChip processing. For each sample, an in-vitro transcription (IVT) reaction was carried out incorporating biotinylated nucleotides according to the manufacturer’s protocol for Illumina® Totalprep RNA amplification kit (Ambion).
|
| |
|
| Hybridization protocol |
1.5µg biotin-labeled cRNA was then hybridized onto Mouse-6 Expression BeadChips (Illumina, San Diego CA) for 16 hours at 55°C. Post-hybridization staining and washing were performed according to manufacturer’s protocols (Illumina).
|
| Scan protocol |
Illumina Sentrix ® Mouse-6 v1.1 BeadChips were scanned using Illumina’s BeadStation 500 scanner.
|
| Description |
We analyzed intensities of 45,905 probes with unique sequences and annotated by Illumina as probes for genomic regions corresponding to protein-coding genes.
|
| Data processing |
Images were checked for grid alignment and then quantified using the BeadStudio software. Control summary graphs generated by BeadStudio were used as quality assurance tools for hybridization, washing stringency, and background. Integrity of the arrays was investigated using the BeadStudio array images and also using bead level image plots generated using the R/beadarray package. Mean pixel intensities by bead type, were created using BeadStudio v3.1 and processed within the R/beadarray package (Dunning et al. 2006).
The raw data conains the log base 2 transformed intensities. A two-step procedure was used for normalization. Quantile normalization (Bolstad et al. 2003) was applied within each tissue type. Then, a correction for batch effects was applied separately for each gene performed using an MM-regression estimator described in Yohai (1987) and computed using the R/robustbase software package (Rousseeuw et al. 2009). The normalized data is also on a log base 2 scale.
|
| |
|
| Submission date |
Feb 01, 2010 |
| Last update date |
Jun 13, 2010 |
| Contact name |
Peter Thomas Vedell |
| E-mail(s) |
vedellpt@gmail.com
|
| Organization name |
The Jackson Laboratory
|
| Street address |
600 Main Street
|
| City |
Bar Harbor |
| State/province |
ME |
| ZIP/Postal code |
04609 |
| Country |
USA |
| |
|
| Platform ID |
GPL6481 |
| Series (1) |
| GSE20121 |
Transcript variation in C57BL/6J mice under normal laboratory conditions |
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