|
| Status |
Public on May 05, 2009 |
| Title |
Thymocytes 24month #3 Cy-3 |
| Sample type |
RNA |
| |
|
| Source name |
Thymocytes 24month #3
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Isolated thymocytes
age: 24 month old
strain: C57BL/6 mouse
|
| Treatment protocol |
Freshly-extracted thymi from mice of various ages were dissociated in RPMI using a syringe and forceps. Cell clumps were broken up with repeated pipetting and then poured through 70um nylon mesh cell strainers (BD Falcon, Bedford, MA) to remove connective tissue and any remaining clumps. The cells were washed once to remove fat cells and red blood cells were lysed with ammonium chloride buffer. The remaining thymocyte population was counted, washed twice in RPMI followed by PBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each array sample, the RNA was prepared from purified thymocytes. The thymocytes were processed by using the RNeasy Mini Kit (Qiagen, Valencia, CA). Quality and quantity of the total RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy3
|
| Label protocol |
total RNA was used to generate fluorescent cRNA for use with Agilent’s oligonucleotide microarrays. The RNA was amplified and labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit following manufactures protocols. In Short: Between 0.5ug to 2ug of total RNA was used to generate first and second strands of cDNA containing a T7 RNA polymerase promoter. Then cRNA was synthesized using T7 RNA polymerase which simultaneously incorporates cyanine 3- or cyanine 5- labeled CTP (Perkin Elmer, Wellesley, MA). Qiagen RNeasy columns (Qiagen Valencia, CA) were used to purify the labeled cRNA and the final concentration was assessed using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies,
|
| |
|
| Hybridization protocol |
750 ng of Cy3-labeled cRNA and 750 ng of Cy5-labeled control sample were combined with spiked in control probes specific for targets on the arrays and hybridized over night at 600C to Agilent Mouse Whole Genome 44K Oligo Microarrays (Agilent Technologies, Palo Alto, CA). The arrays were washed at room temperature 6X SSC with 0.005% Triton X-102 for 10 minutes and 0.01x SSC with 0.005% Triton X-102 at 4 degrees C for 5 minutes. The slides were then dried in a nitrogen stream.
|
| Scan protocol |
Arrays were scanned at a 10um resolution using an Agilent Microarray Scanner, G2565AA .
|
| Description |
Isolated thymocytes from a 24 month old C57BL/6 mouse
Test channel of GSM400017
|
| Data processing |
Data from both the Cy-3 and the Cy5 channels was extracted using the Agilent Feature Extraction Software, Version 7.1. Results for control probes were first removed, multiple repeats of any probe were averaged, then each array was z-normalized. In short, the natural log of the remaining genes were used to find the avg and std of each array and the z-score normalization was calculated: Z-score = (raw value - avg)/std. Results for the Cy-3 and Cy-5 channels will be reported separately. In order to be selected for the final gene list, the expression value of a particular gene had to be at least 1.5 times different from the Z score of the control. Differences were considered statistically significant only if they had a p value less than 0.01. Complete data on all spots will be included in the supplemental files.
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| |
|
| Submission date |
May 04, 2009 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
| GSE15945 |
Transcriptome analysis of murine thymocytes reveals age-associated changes in thymic gene expression |
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