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Sample GSM1255182 Query DataSets for GSM1255182
Status Public on Feb 12, 2015
Title PC-1d-C3
Sample type RNA
 
Source name LCM Crypt fraction of Proximal colon after CONV-D day 1
Organism Mus musculus
Characteristics strain: C57Bl/6
gender: female
age: 10-12 weeks
bacterial status: CONV-D day 1
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_1d-PC-3-Crypt_FS324-18_130418.CEL
Gene expression data from LCM Crypt fraction of Proximal colon after CONV-D day 1 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

Data table header descriptions
ID_REF
VALUE log2 of processed signal

Data table
ID_REF VALUE
17210850 5.74123
17210852 6.062624
17210855 10.54808
17210869 9.849197
17210883 5.910775
17210887 8.179065
17210904 6.243052
17210912 8.256227
17210947 6.329229
17210953 6.038443
17210984 9.039132
17210994 6.006547
17210996 5.743012
17210998 5.604299
17211000 7.838668
17211004 6.332414
17211023 5.961376
17211033 6.729351
17211043 6.695004
17211066 5.76006

Total number of rows: 41345

Table truncated, full table size 722 Kbytes.




Supplementary file Size Download File type/resource
GSM1255182_PA181_1d-PC-3-Crypt_FS324-18_130418_MoGene-2_0-st_.CEL.gz 8.4 Mb (ftp)(http) CEL
Processed data included within Sample table

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