|
| Status |
Public on May 02, 2007 |
| Title |
P-HT-192-2-260 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
hypothalamus (192 h after injection of LL/2(LLC1) lung tumor cells)
|
| Organism |
Mus musculus |
| Characteristics |
Hypothalamus form male C57BL/6 mice 192 h after s.c. injection of LL/2(LLC1) lung tumor cells
|
| Treatment protocol |
Eight week old C57Bl/6 male mice were injected s.c. with 1E6 LL/2(LLC1) lung tumor cells in 300 ul PBS and sacrifice by decapitation after 192 h
|
| Growth protocol |
Mice were housed in HEPA filtered air racks (Tecniplast, Italy) with food and water ad libitum
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol Reagent (Invitrogen). Poly A+ RNA was obtained from total RNA using the MicroPoly(A) Pure kit (Ambion)
|
| Label |
Cy3
|
| Label protocol |
RNA was reversed transcribed using Superscript II RT (Invitrogen) with oligo dT primers and random primers, both in the presence of aa-dUTP (Sigma). The cDNA was labeled with Cy3 or Cy5 dyes (Amersham), resuspended in DMSO, and incubated for 1 hr at room temperature in the dark. The probes were purified using the SNAP Gel Purification Kit (Invitrogen) following manufacturer instructions with the following modification: at the initial step, 500 ul of loading buffer (2.25 M guanidinium HCl in 70% isopropanol) were added to the sample, placed in a SNAP column and incubated for 4 min before the first centrifugation
|
| |
|
| Channel 2 |
| Source name |
hypothalamus (control)
|
| Organism |
Mus musculus |
| Characteristics |
Hypothalamus from male C57Bl/6 mice 192 h after s.c. injection of PBS
|
| Treatment protocol |
Eight week old C57Bl/6 male mice were injected s.c. with 300 ul PBS and sacrifice by decapitation after 192 h
|
| Growth protocol |
Mice were housed in HEPA filtered air racks (Tecniplast, Italy) with food and water ad libitum
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol Reagent (Invitrogen). Poly A+ RNA was obtained from total RNA using the MicroPoly(A) Pure kit (Ambion)
|
| Label |
Cy5
|
| Label protocol |
RNA was reversed transcribed using Superscript II RT (Invitrogen) with oligo dT primers and random primers, both in the presence of aa-dUTP (Sigma). The cDNA was labeled with Cy3 or Cy5 dyes (Amersham), resuspended in DMSO, and incubated for 1 hr at room temperature in the dark. The probes were purified using the SNAP Gel Purification Kit (Invitrogen) following manufacturer instructions with the following modification: at the initial step, 500 ul of loading buffer (2.25 M guanidinium HCl in 70% isopropanol) were added to the sample, placed in a SNAP column and incubated for 4 min before the first centrifugation
|
| |
|
| |
| Hybridization protocol |
Slides were prehybridised at 42°C in 2X SSC, 0.1% SDS, 1% BSA. Labeled probes were mixed with hybridization buffer containing 30% formamide and hybridized overnight at 42°C
|
| Scan protocol |
Images were obtained with a VersArray ChipReader system (BioRad) at 10 um resolution and 16bit depth. Each slide was scanned three times, each time with a 5% increment in the sensitivity of the scanner. The data integration for multipole scanning were calculated by M = log2(cy3 / cy2) and A = 0.5 * log2(cy2 * cy3) values for all the possible combinations between channels among the three scannings. Quality score for each couple of channels was calculated as QS = min(QScy2, QScy3). The resulting 9 sets of A and M values were averaged using the QS as weights to obtain the integrated A and M sets.
|
| Description |
hypothalamus of male C57Bl/6 mice
|
| Data processing |
Data was log2 transformed and normalizaed with an intensity- and location-dependent loess fit
|
| |
|
| Submission date |
Mar 14, 2007 |
| Last update date |
May 04, 2007 |
| Contact name |
Mariano Cesar Salibe |
| E-mail(s) |
msalibe@leloir.org.ar
|
| Organization name |
Gentron LLC
|
| Street address |
Av. Patricias Argentinas 435
|
| City |
Ciudad de Buenos Aires |
| ZIP/Postal code |
1425 |
| Country |
Argentina |
| |
|
| Platform ID |
GPL4983 |
| Series (2) |
| GSE7295 |
profiling of hypothalamus from mice s.c. injected with LL/2(LLC1) lung carcinoma cells vs. PBS |
| GSE7565 |
Profiling brain and liver tissues from mice |
|
