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Sample GSM364904 Query DataSets for GSM364904
Status Public on Apr 03, 2009
Title cultured_crypt-organoid_vs_small_intestinal_crypt_ 1
Sample type RNA
 
Channel 1
Source name cells from cultured crypt organoids
Organism Mus musculus
Characteristics cells isolated form cultured crypt-organoids grown in crypt culture medium (Advanced DMEM/F12 with growth factors (10-50 ng/ml EGF (Peprotech), 500 ng/ml R-spondin 111 and 100 ng/ml Noggin (Peprotech))
Treatment protocol Isolated small intestines were opened longitudinally, and washed with cold PBS. The tissue was chopped into around 5 mm pieces, and further washed with cold PBS. The tissue fragments were incubated in 2 mM EDTA with PBS for 30 min on ice. After removal of EDTA medium, the tissue fragments were vigorously suspended by 10 ml pipette with cold PBS. The supernatant was the villous fraction and was discarded; the sediment was resuspended with PBS. After further vigorous suspension and centrifugation, the supernatant was enriched for crypts. This fraction was passed through a 70-um cell strainer (BD bioscience) to remove residual villous material. Isolated crypts were centrifuged at 300 rpm for 3 min to separate crypts from single cells. The final fraction consisted of essentially pure crypts and was used for culture or direct RNA isolation.
Growth protocol Isolated crypts were counted and pelleted. 500 crypts were mixed with 50 ul Matrigel (BD Bioscience) and plated in 24 well plates. After polymerization of Matrigel, 500 ul of crypt culture medium (Advanced DMEM/F12 with growth factors (10-50 ng/ml EGF (Peprotech), 500 ng/ml R-spondin 111 and 100 ng/ml Noggin (Peprotech)) was added. For passage, organoids were removed from Matrigel and mechanically dissociated into single-crypt domains, and transferred to new Matrigel. Passage was performed every 1-2 weeks with 1:5 split ratio.
Extracted molecule total RNA
Extraction protocol RNA was isolated using Trizol (Invitrogen)
Label Cy3
Label protocol 500 ng of total RNA was labeled using low RNA Input Linear Amp kit (Agilent Technologies, Pato Alto, CA, USA).
 
Channel 2
Source name epithelial cells from small intestine crypts
Organism Mus musculus
Characteristics epithelial cells from small intestine crypts isolated by EDTA and separated from villus by a cell strainer
Treatment protocol Isolated small intestines were opened longitudinally, and washed with cold PBS. The tissue was chopped into around 5 mm pieces, and further washed with cold PBS. The tissue fragments were incubated in 2 mM EDTA with PBS for 30 min on ice. After removal of EDTA medium, the tissue fragments were vigorously suspended by 10 ml pipette with cold PBS. The supernatant was the villous fraction and was discarded; the sediment was resuspended with PBS. After further vigorous suspension and centrifugation, the supernatant was enriched for crypts. This fraction was passed through a 70-um cell strainer (BD bioscience) to remove residual villous material. Isolated crypts were centrifuged at 300 rpm for 3 min to separate crypts from single cells. The final fraction consisted of essentially pure crypts and was used for culture or direct RNA isolation.
Growth protocol Isolated crypts were counted and pelleted. 500 crypts were mixed with 50 ul Matrigel (BD Bioscience) and plated in 24 well plates. After polymerization of Matrigel, 500 ul of crypt culture medium (Advanced DMEM/F12 with growth factors (10-50 ng/ml EGF (Peprotech), 500 ng/ml R-spondin 111 and 100 ng/ml Noggin (Peprotech)) was added. For passage, organoids were removed from Matrigel and mechanically dissociated into single-crypt domains, and transferred to new Matrigel. Passage was performed every 1-2 weeks with 1:5 split ratio.
Extracted molecule total RNA
Extraction protocol RNA was isolated using Trizol (Invitrogen)
Label Cy5
Label protocol 500 ng of total RNA was labeled using low RNA Input Linear Amp kit (Agilent Technologies, Pato Alto, CA, USA).
 
 
Hybridization protocol according to Agilent guidelines
Scan protocol according to Agilent guidelines
Description crypt vs. crypt-organoid 1 dyeswap
Data processing Microarray signal and background information were retrieved using Feature Extraction (V.9.5.3, Agilent Technologies). All data analyses were performed using ArrayAssist (5.5.1, Stratagene Inc.) and Microsoft Excel (Microsoft Corporation). Raw signal intensities were corrected by subtracting local background. Negative values were changed into a positive value close to zero (standard deviation of the local background) in order to allow calculation of ratios between intensities for features only present in one channel (small intestinal crypts or organoids). Normalization was performed by applying a Lowess algorithm and individual features were filtered if both (small intestinal crypts or organoids) intensities were changed or if both intensities were less than two times the background signal. Furthermore, non-uniform features were filtered.
 
Submission date Jan 27, 2009
Last update date Apr 03, 2009
Contact name Daniel E. Stange
E-mail(s) daniel.stange@uniklinikum-dresden.de
Organization name University Clinic Dresden
Street address Fetscherstr. 74
City Dresden
ZIP/Postal code 01307
Country Germany
 
Platform ID GPL7202
Series (1)
GSE14594 Single intestinal stem cells build crypt-villus structures in vitro without a cellular niche

Data table header descriptions
ID_REF
VALUE normalized log2 ratio Cy3/Cy5
INV_VALUE normalized log2 ratio Cy5/Cy3

Data table
ID_REF VALUE INV_VALUE
A_52_P756921 0 0
A_52_P1133703 0.556308 -0.55630773
A_52_P1052870 -0.155933 0.15593274
A_52_P1021909 0.186575 -0.18657462
A_51_P133060 -2.52035 2.5203452
A_51_P287221 0.295503 -0.29550257
A_52_P112159 0.405972 -0.405972
A_51_P378063 2.5693 -2.5693026
A_52_P289204 -0.710293 0.71029305
A_51_P439156 -0.599385 0.59938526
A_52_P2181 -0.33916 0.33916005
A_52_P53154 0 0
A_52_P527925 -0.0441429 0.044142872
A_52_P315910 0.284732 -0.2847323
A_52_P519870 0.109399 -0.10939931
A_52_P96087 -0.00701224 0.007012236
A_51_P137808 -0.802283 0.8022832
A_52_P552026 -0.0688296 0.06882958
A_52_P42395 -0.229327 0.22932653
A_52_P350876 -0.864067 0.8640675

Total number of rows: 41174

Table truncated, full table size 1242 Kbytes.




Supplementary file Size Download File type/resource
GSM364904.txt.gz 15.8 Mb (ftp)(http) TXT
Processed data included within Sample table

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