|
| Status |
Public on Sep 01, 2008 |
| Title |
K14E6_cervix_Technical_replica |
| Sample type |
RNA |
| |
|
| Source name |
K14E6_cervix and vagina_pool of 3 mice
|
| Organism |
Mus musculus |
| Characteristics |
K14E6 transgenic 6-week-old virgin female mice were employed. All mice were sacrificed and shaved with razor. After sacrifice lower reproductive tract (cervix and vagina) were removed. and immediately frozen in liquid nitrogen for later RNA isolation.
|
| Treatment protocol |
Tissue was immediately frozen in liquid nitrogen for later RNA isolation using Trizol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Collected tissues were homogenized by mortar in liquid nitrogen, total RNA was extracted using TRIZOL reagent (InVitrogen) and purified using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
The quality and size distribution of the RNA were assessed with the RNA Nano Lab on a Chip kit (Agilent Technologies), which yielded RNA integrity numbers (RIN) from 6.5 to 9.2. Total RNA collected from skin or cervical tissue from three female mice of each condition were pooled. Briefly, 3 μg of the total pooled RNA was converted to first-strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer. Second strand cDNA synthesis was followed by an in vitro transcription reaction in which biotinylated CTP and UTP were incorporated to the generated transcripts.
|
| |
|
| Hybridization protocol |
The cRNA products were fragmented to 200 nucleotides or less, then, 15 μg of the fragmentation product were used to prepare 300 μl hybridization cocktail (100mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween-20, 0.1 mg ml-1 of HS DNA, and 0.5 mg ml-1 acetylated bovine serum albumine). The cocktails were heated to 95°C and hybridized in the Mouse Genome 430A 2.0 Array (Affymetrix Inc.) for 16 hours at 45ºC .After hybridization, arrays were washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
The GeneChip Scanner 3000 7G (Affymetrix, Santa Clara CA) was used to collect fluorescence signal of 11um feature size resolution after excitation at 570 nm. GCOS software (Affymetrix, Santa Clara CA) was used to obtain intensity signal and quality data of the scanned arrays.
|
| Description |
Pooled total RNA from three transgenic mice
|
| Data processing |
Chip scanning and expression analyisis of the .CHP files was performed using the MAS 5.0 software
|
| |
|
| Submission date |
Mar 03, 2008 |
| Last update date |
Mar 18, 2009 |
| Contact name |
Alfredo Hidalgo-Miranda |
| E-mail(s) |
[email protected]
|
| Phone |
+52-55-53501966
|
| Organization name |
National Institute of Genomic Medicine
|
| Street address |
Periferico Sur 4124, Torre Zafiro II, piso 6
|
| City |
Mexico City |
| ZIP/Postal code |
01900 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
| GSE10702 |
Gene expression profile of cervical and skin tissues from HPV 16 E6 transgenic mice |
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