|
| Status |
Public on Feb 12, 2015 |
| Title |
PC-5d-C3 |
| Sample type |
RNA |
| |
|
| Source name |
LCM Crypt fraction of Proximal colon after CONV-D day 5
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
gender: female
age: 10-12 weeks
bacterial status: CONV-D day 5
tissue: Proximal colon
fraction: Crypt
replicate: 3
|
| Treatment protocol |
10-12 weeks old mice were sacrificed before and during the time course of colonization with a normal microbiota (on days 1, 3, 5 and 7) by cervical dislocation and the intestine removed. The distal part of the small intestine (ileum) and the proximal part of the colon were excised, flushed with PBS and finally flushed and embedded with OCT freezing medium. The complete procedure from sacrificing the mouse until the frozen tissue blocks took less than five minutes to preserve RNA integrity. All material and solutions used further were RNase-free. 8 µm thick cryosections were cut from the OCT blocks at -20°C using a microtome (Leica), placed onto PEN membrane slides (ZEISS) and immediately stained for LCM. Briefly, slides were dehydrated in absolute and 70% ethanol for 30s each, dipped into RNase-free water to remove excessive OCT and then incubated in 1% cresyl violet in 50% ethanol step for another 30s. Slides were partially destained by dipping in 70% and absolute ethanol before air-drying. Slides were stored in airtight containers at -80°C until laser capture microdissection (LCM). Airtight containers were equilibrated to ambient temperature before opening. LCM was performed using PALM Microbeam Microdissection microscope (ZEISS) and fractions were collected dry. The harvested fractions were immediately lyzed in RLT containing 1% β-mercaptoethanol and stored at -80°C.
|
| Growth protocol |
Germ-free C57Bl/6 mice were maintained in standard makrolon cages according to standard protocols inside sterile flexible film isolators. Conventionally raised C57Bl/6 mice were kept in sterilized, filter-topped IVC cages under specific pathogen-free conditions. All animals were housed with wood shavings and wooden wool and had free access to standard autoclaved chow diet (Labdiet, USA) and water. Mice were kept with 12 hours light/dark cycle at 18-22°C temperature and 35-65% relative humidity which was continuously and automatically registered and regulated. A maximum of 5 mice were kept in one cage. Germ-free mice were colonized by oral gavage of pooled caecal content of conventionally raised age and sex matched mice. Animals used for experiments were females and 10-12 weeks of age at harvest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from the lysates of the LCM harvested fractions using RNeasy Micro Kit (Qiagen, Hilden, Germany). RNA concentration and quality were evaluated using capillary electrophoresis on a 2100 Bioanalyzer with RNA 6000 Pico kit (Agilent Technologies, Santa Clara, CA, USA).
|
| Label |
biotin
|
| Label protocol |
1000 picograms of total RNA from each sample were used to generate amplified and biotinylated sense transcript cDNA from the entire expressed transcriptome according to the Nugen Technologies, Inc. protocols Ovation® Pico WTA System V2 (M01224v2) and Encore Biotine Module (M01111v5).
|
| |
|
| Hybridization protocol |
GeneChip® ST Arrays (GeneChip® Mouse Gene 2.0 ST Array) were hybridized for 16 hours in a 45°C incubator, rotated at 60 rpm. According to the GeneChip® Expression Wash, Stain and Scan Manual (PN 702731 Rev 3, Affymetrix Inc., Santa Clara, CA) the arrays were then washed and stained using the Fluidics Station 450.
|
| Scan protocol |
Arrays were scanned using the GeneChip® Scanner 3000 7G (Affymetrix).
|
| Description |
PA181_5d-PC-3-Crypt_FS324-42_130418.CEL
Gene expression data from LCM Crypt fraction of Proximal colon after CONV-D day 5 of 10-12 weeks old female C57Bl/6 mouse, biological replicate #3
|
| Data processing |
The data was processed with quantile normlaization, pm-only for background coreection, iterative PLIER algorithm for expresion index calculation. The analysis was performed by expression console software
|
| |
|
| Submission date |
Oct 30, 2013 |
| Last update date |
Feb 13, 2015 |
| Contact name |
Intawat Nookaew |
| E-mail(s) |
inookaew@uams.edu
|
| Organization name |
University of Arkansas for Medical Sciences
|
| Department |
Department of Biomedical Informatics
|
| Street address |
4301 W Markham St
|
| City |
Little Rock |
| ZIP/Postal code |
72205 |
| Country |
USA |
| |
|
| Platform ID |
GPL16570 |
| Series (2) |
| GSE51911 |
Whole transcriptome analysis of laser capture microdissected tissues reveals site-specific programming of the host epithelial transcriptome by the gut microbiota [short period of bacteria colonization] |
| GSE51912 |
Whole transcriptome analysis of laser capture microdissected tissues reveals site-specific programming of the host epithelial transcriptome by the gut microbiota |
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