|
| Status |
Public on Sep 01, 2009 |
| Title |
VLcmT clone 2, bio rep 2, TAg Cy5/wt Cy3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
VLcmT2 (Cy5)
|
| Organism |
Mus musculus |
| Characteristics |
villi enterocytes captured by LCM (VLcm) of TAg transgenic mice
|
| Growth protocol |
All mice were maintained in an animal facility without restriction to food or water. Mice were sacrificed and their entire small intestine was removed, opened longitudinally and washed in saline solution. The middle 1/3 of the small intestine (“whole intestine”) was harvested and whole intestine rolls were prepared. Rolls were processed to isolate villi cell types by laser capture microscopy (LCM). Briefly, the rolls were quickly frozen and stored at -80°C. Frozen sections were cut and immediately dehydrated in graded alcohol solutions. The dried sections were examined microscopically and the top 2/3rds of 50 villous segments from each section were collected using a laser capture microscope (Pix Cell II, Arcturus, Mt. View, CA). The captured tissue (VLcm) was extracted in Pico Pure extraction buffer (Arcturus) according to the manufacturer’s directions and stored at -80°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with the PicoPure kit (Arcturus) and amplified twice with the RiboAmp kit (Arcturus).
|
| Label |
Cy5
|
| Label protocol |
Total RNA was isolated and converted to Cy3- and Cy5-labeled cDNA in independent reactions. 15ug of total RNA was placed in a reaction that contained 7.5ug of Random Primers in a total volume of 15.4ul and placed at 70°C for 10min. After chilling the reactions on ice, labeled cDNA was prepared by adding 3ul of 1mM Cy3-dUTP or 1mM Cy5-dUTP and 11.6ul of a reverse transcription cocktail and the reaction was incubated at 42°C for 2 hours. The final concentration of the reagents were 13.3 U/ul SuperScript II Reverse Transcriptase, 1X first strand buffer, 10mM DTT, 0.5 mM each dATP, dCTP, dGTP and 0.2mM of dTTP. RNA was degraded by adding 1.5ul of 1N NaOH and 1.0ul of 60mM EDTA to each reaction and incubated at 65°C for 10 minutes. 470ul of TE pH 7.4 was added to the reaction and applied to a Microcon-30 filter. The reactions were centrifuged for 7 min at 8,500g and washed once with 400ul of TE pH7.4 to remove unincorporated dyes. Filters were centrifuged upside-down to harvest the labeled cDNA (~50ul). Next, a cDNA labeled with Cy3 was mixed with a cDNA labeled with Cy5 and purified using the QIAQuick Kit. After completely drying the cDNA in a vacuum centrifuge, the labeled cDNA was resuspended in 7.5ul of dH2O.
|
| |
|
| Channel 2 |
| Source name |
VLcm.wt2 (Cy3)
|
| Organism |
Mus musculus |
| Characteristics |
villi enterocytes captured by LCM (VLcm) of wild-type
|
| Growth protocol |
All mice were maintained in an animal facility without restriction to food or water. Mice were sacrificed and their entire small intestine was removed, opened longitudinally and washed in saline solution. The middle 1/3 of the small intestine (“whole intestine”) was harvested and whole intestine rolls were prepared. Rolls were processed to isolate villi cell types by laser capture microscopy (LCM). Briefly, the rolls were quickly frozen and stored at -80°C. Frozen sections were cut and immediately dehydrated in graded alcohol solutions. The dried sections were examined microscopically and the top 2/3rds of 50 villous segments from each section were collected using a laser capture microscope (Pix Cell II, Arcturus, Mt. View, CA). The captured tissue (VLcm) was extracted in Pico Pure extraction buffer (Arcturus) according to the manufacturer’s directions and stored at -80°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with the PicoPure kit (Arcturus) and amplified twice with the RiboAmp kit (Arcturus).
|
| Label |
Cy3
|
| Label protocol |
Total RNA was isolated and converted to Cy3- and Cy5-labeled cDNA in independent reactions. 15ug of total RNA was placed in a reaction that contained 7.5ug of Random Primers in a total volume of 15.4ul and placed at 70°C for 10min. After chilling the reactions on ice, labeled cDNA was prepared by adding 3ul of 1mM Cy3-dUTP or 1mM Cy5-dUTP and 11.6ul of a reverse transcription cocktail and the reaction was incubated at 42°C for 2 hours. The final concentration of the reagents were 13.3 U/ul SuperScript II Reverse Transcriptase, 1X first strand buffer, 10mM DTT, 0.5 mM each dATP, dCTP, dGTP and 0.2mM of dTTP. RNA was degraded by adding 1.5ul of 1N NaOH and 1.0ul of 60mM EDTA to each reaction and incubated at 65°C for 10 minutes. 470ul of TE pH 7.4 was added to the reaction and applied to a Microcon-30 filter. The reactions were centrifuged for 7 min at 8,500g and washed once with 400ul of TE pH7.4 to remove unincorporated dyes. Filters were centrifuged upside-down to harvest the labeled cDNA (~50ul). Next, a cDNA labeled with Cy3 was mixed with a cDNA labeled with Cy5 and purified using the QIAQuick Kit. After completely drying the cDNA in a vacuum centrifuge, the labeled cDNA was resuspended in 7.5ul of dH2O.
|
| |
|
| |
| Hybridization protocol |
The cDNA was applied to an Agilent Mouse cDNA microarray (Agilent Technologies, G4104A) for 17 hours at 65°C. The array was washed and dried as recommended by the manufacturer.
|
| Scan protocol |
Using an Affymetrix GMS418 scanner, two images from each array were extracted by exciting the Cy3 dye at 532nm and the Cy5 dye at 635nm and saved as a 16-bit TIFF file. The Cy3 and Cy5 image were aligned and quantitated with Imagene 4.2 (Biodiscovery). The entire array was scanned manually to identify and flag any spots that were masked by debris or scratches.
|
| Description |
VLcmT2 technical replicate 1
|
| Data processing |
We flagged any spots whose Signal Mean was not greater than the Background Median plus two times the Background Standard Deviation. Also, all control features on each array were flagged. Each array also contained a set of features that were flagged by Agilent. All unflagged spots were background subtracted by taking the difference of the Signal Mean and the Background Median. The background subtracted signals were normalized with a lowess intensity-dependent smoother using BRB-Array Tools (Rich Simon, National Cancer Institute) except that the lowess span parameter was changed from 0.4 to 0.2. A log ratio was calculated for each biological replicate by averaging its two dye-flip log ratios.
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| |
|
| Submission date |
May 24, 2007 |
| Last update date |
May 29, 2009 |
| Contact name |
Daniel Clark |
| Organization name |
University of Pittsburgh
|
| Street address |
3501 Terrace St.
|
| City |
Pittsburgh |
| ZIP/Postal code |
15213 |
| Country |
USA |
| |
|
| Platform ID |
GPL872 |
| Series (2) |
| GSE7892 |
Global analysis of gene expression in MEF cells and villi enterocytes expressing SV40 T antigens |
| GSE7906 |
Cell-type Specific Regulation of Gene Expression by Simian Virus 40 T antigens |
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