|
| Status |
Public on Jun 28, 2012 |
| Title |
PAX8_vs_scramble_2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
PCCL3 PAX silenced
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell type: PCCl3 thyroid cells
|
| Treatment protocol |
PCCl3 cells transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) both for scramble and PAX8 siRNAs conditions (10 ng siRNA /ml) (Dharmacon, Denver, USA).
|
| Growth protocol |
PCCl3 thyroid cells were grown were grown in Coon’s modified Ham’s F-12 medium supplemented with 5% donor calf serum and a six-hormone mixture [1 nM TSH, 10 g/ml insulin, 10 ng/ml somatostatin, 5 g/ml transferrin, 10 nM hydrocortisone, and 10 ng/ml glycyl-L-histidyl-L-lysine acetate]
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation was performed using TRIzol reagent (Invitrogen, Carlsbad, CA), following the manufacturer’s recommended protocol
|
| Label |
Cy3
|
| Label protocol |
Labelling was performed by using the Low RNA Input Linear Amplification Kit, PLUS, Two-Color (Agilent Technologies, Palo Alto, CA). Briefly, for each sample 2 μg of total RNA input were amplified in two rounds of amplification by following manufacturer’s instructions. The first strand cDNA synthesis and amplification reactions were carried out by using random and T7 primers, respectively. During the 2 hour in vitro transcription, Cy3- or Cy5-labeled CTP was incorporated on each amplified RNA (cRNA). Products of the reaction were then purified using RNAeasy mini spin column.
|
| |
|
| Channel 2 |
| Source name |
PCCL3_scramble
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell type: PCCl3 thyroid cells
|
| Treatment protocol |
PCCl3 cells transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) both for scramble and PAX8 siRNAs conditions (10 ng siRNA /ml) (Dharmacon, Denver, USA).
|
| Growth protocol |
PCCl3 thyroid cells were grown were grown in Coon’s modified Ham’s F-12 medium supplemented with 5% donor calf serum and a six-hormone mixture [1 nM TSH, 10 g/ml insulin, 10 ng/ml somatostatin, 5 g/ml transferrin, 10 nM hydrocortisone, and 10 ng/ml glycyl-L-histidyl-L-lysine acetate]
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation was performed using TRIzol reagent (Invitrogen, Carlsbad, CA), following the manufacturer’s recommended protocol
|
| Label |
Cy5
|
| Label protocol |
Labelling was performed by using the Low RNA Input Linear Amplification Kit, PLUS, Two-Color (Agilent Technologies, Palo Alto, CA). Briefly, for each sample 2 μg of total RNA input were amplified in two rounds of amplification by following manufacturer’s instructions. The first strand cDNA synthesis and amplification reactions were carried out by using random and T7 primers, respectively. During the 2 hour in vitro transcription, Cy3- or Cy5-labeled CTP was incorporated on each amplified RNA (cRNA). Products of the reaction were then purified using RNAeasy mini spin column.
|
| |
|
| |
| Hybridization protocol |
All procedures of hybridization, slide and image processing were carried out according to the manufacturer’s instructions (Two-Color Microarray-Based Gene Expression Analysis protocol). In each experiment, 825 ng of contrasting cRNA samples were fragmented at 60°C for 30 min and hybridized at 65ºC for 17 h.
|
| Scan protocol |
Agilent G2565BA Microarray Scanner
Signal quantification was carried out with Feature Extraction 9.1 software
|
| Description |
Biological Replicate
|
| Data processing |
Limma package for R was used for background subtraction and LOWESS and QUANTILE normalization.
|
| |
|
| Submission date |
Jan 28, 2011 |
| Last update date |
Jun 28, 2012 |
| Contact name |
Pilar Santisteban |
| E-mail(s) |
psantisteban@iib.uam.es
|
| Phone |
+44 915854455
|
| URL |
http://www.iib.uam.es
|
| Organization name |
Biomedical Research Institute-CSIC
|
| Department |
Physiopathology of the Endocrine and Nervous System
|
| Lab |
2.9_Thyroid Transcripcion
|
| Street address |
Arturo Duperier 4
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4135 |
| Series (2) |
| GSE26937 |
PAX8 transcriptional profiling in rat PCCL3 cells |
| GSE26938 |
Global analysis of in vivo Pax8-binding sites in rat thyroid cells using high-throughput technologies: Chip-Seq and whole genome expression arrays |
|
