|
| Status |
Public on Apr 03, 2009 |
| Title |
small_intestinal_crypt_vs_cultured_crypt-organoid_2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| 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 |
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 |
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 |
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 2
|
| 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) |
[email protected]
|
| 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 |
|
