|
| Status |
Public on Mar 31, 2014 |
| Title |
4thMammaryGlands_ContolateralVsCleared_d19_2BRs |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cleared vs controlateral 4th mammary gland, d19 of pregnancy
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: CD1
tissue: 4th mammary glands
developmental stage: d19 of pregnancy
|
| Treatment protocol |
For the study 60 female CD1 mice obtained by in-house breeding. At the weaning age (3 weeks old) they were subjected to the removing of the epithelial component of the 4th right mammary gland in order to obtain a cleared fat pad. The 4th left gland of the same animal underwent a simple skin excision for control evaluations. The success of the clearing procedure was assessed by whole-mount analysis (Rasmussen SB et al. 2000) revealing the presence of the entire ductal tree in the removed part of mammary fat pad. At the adult stage (10-15 weeks of age), mice that underwent appropriate epithelial clearing of the mammary gland were mated and subsequently sacrificed on pregnancy day 0, 10, 15, 17 and 19 (6 mice/group) to collect tissue samples from cleared and control mammary glands. Collected specimens were appropriately sampled for RNA extraction and microarray analysis and eventual morphological examination.
|
| Growth protocol |
Animal experimentation was performed using CD1 mice housed in the Animal Facility of the Polytechnic University of Marche. Animals were fed a standard chow diet and they were kept at 12hr light/12hr dark cycle. Animal care and handling were performed in accordance with the Italian Institutional Guidelines, and the experimental protocol was approved by the local Ethical Committee for Animal Experimentation For the study 60 female CD1 mice were used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation using TriZol (see supplementary file for details)
|
| Label |
Cy3,Cy5
|
| Label protocol |
labeling of eukaryotic total RNA with aminoallyl labeled nucleotides via first strand cDNA synthesis followed by a coupling of the aminoallyl groups to either Cyanine 3 or 5 (Cy 3/Cy5) fluorescent molecules (see supplementary file for details)
|
| |
|
| Channel 2 |
| Source name |
Controlateral vs cleared 4th mammary gland, d19 of pregnancy
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: CD1
tissue: 4th mammary glands
developmental stage: d19 of pregnancy
|
| Treatment protocol |
For the study 60 female CD1 mice obtained by in-house breeding. At the weaning age (3 weeks old) they were subjected to the removing of the epithelial component of the 4th right mammary gland in order to obtain a cleared fat pad. The 4th left gland of the same animal underwent a simple skin excision for control evaluations. The success of the clearing procedure was assessed by whole-mount analysis (Rasmussen SB et al. 2000) revealing the presence of the entire ductal tree in the removed part of mammary fat pad. At the adult stage (10-15 weeks of age), mice that underwent appropriate epithelial clearing of the mammary gland were mated and subsequently sacrificed on pregnancy day 0, 10, 15, 17 and 19 (6 mice/group) to collect tissue samples from cleared and control mammary glands. Collected specimens were appropriately sampled for RNA extraction and microarray analysis and eventual morphological examination.
|
| Growth protocol |
Animal experimentation was performed using CD1 mice housed in the Animal Facility of the Polytechnic University of Marche. Animals were fed a standard chow diet and they were kept at 12hr light/12hr dark cycle. Animal care and handling were performed in accordance with the Italian Institutional Guidelines, and the experimental protocol was approved by the local Ethical Committee for Animal Experimentation For the study 60 female CD1 mice were used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation using TriZol (see supplementary file for details)
|
| Label |
Cy5,Cy3
|
| Label protocol |
labeling of eukaryotic total RNA with aminoallyl labeled nucleotides via first strand cDNA synthesis followed by a coupling of the aminoallyl groups to either Cyanine 3 or 5 (Cy 3/Cy5) fluorescent molecules (see supplementary file for details)
|
| |
|
| |
| Hybridization protocol |
hybridization of a Cy labeled cDNA probe (mix of Cy3 and Cy5) onto epoxy coated slide spotted with PCR products (see supplementary file for details)
|
| Scan protocol |
Axon 4000B scanner was used with GenePix 5 software.
|
| Description |
2 biological replicates available, dye swap for 1
|
| Data processing |
Spots showing low intensity, inhomogeneity, saturation and too small diameters were filtered out. In order to increase the dynamic range of the system all slides were scanned with two photo multiplier tube voltage settings. An algorithm was implemented in Perl which supplements the saturated spots in the high intensity scan with the corresponding values from the low intensity scan multiplied with a correction factor (Lyng et al., 2004). Normalization was done with the help of the software Carmaweb (Rainer J et al., 2006). Parameters:
1) Pintip Loess Normalization
2) Normalization of dyeswap pairs
3) Log2 transformation of resulting ratios
|
| |
|
| Submission date |
Aug 29, 2013 |
| Last update date |
Mar 31, 2014 |
| Contact name |
Andreas Prokesch |
| E-mail(s) |
[email protected]
|
| Organization name |
Medical University Graz
|
| Department |
Institute of Cell Biology, Histology, and Embryology
|
| Street address |
Harrachgasse 21/7
|
| City |
Graz |
| State/province |
Styria |
| ZIP/Postal code |
8010 |
| Country |
Austria |
| |
|
| Platform ID |
GPL17656 |
| Series (1) |
| GSE50447 |
Mammary development during pregnancy (controlateral control vs. cleared fat pad) to define transdifferentiation factors |
|
