|
| Status |
Public on Jan 01, 2010 |
| Title |
PNRA830238G |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
normal protein, 28 days old
|
| Organism |
Sus scrofa |
| Characteristics |
deathweight: 5.102
birthweight: 0.88
sex: Male
diet: normal protein
site: distal small intestine
age: 28 days
batch: IUGR
birth date: 23/01/2008
pair: 11
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from individual frozen samples of distal small intestine and colonic segments collected from each pig, as previously described (Chomczynski and Sacchi 1987). RNA was quantified by absorbance at 260 nm (Multiskan Spectrum, Thermo Labsystems) and its integrity was estimated using RNA 6000 nano (LabChip® kit in 2100 Bioanalyser, Agilent, France).
|
| Label |
Cy5
|
| Label protocol |
Five micrograms of RNA samples were labelled with Cy5 and hybridized against a common reference (5 microg of a mix of all RNA samples used in the experiment) labelled with Cy3 using the ChipShotTM Direct Labeling and Clean-Up system (Promega) according to the manufacturer instructions for total RNA labeling.
|
| |
|
| Channel 2 |
| Source name |
Pool of equal amount of all tissue with prefix=PNRA
|
| Organism |
Sus scrofa |
| Characteristics |
number of samples in the pool: 22
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from individual frozen samples of distal small intestine and colonic segments collected from each pig, as previously described (Chomczynski and Sacchi 1987). RNA was quantified by absorbance at 260 nm (Multiskan Spectrum, Thermo Labsystems) and its integrity was estimated using RNA 6000 nano (LabChip® kit in 2100 Bioanalyser, Agilent, France).
|
| Label |
Cy3
|
| Label protocol |
Five micrograms of RNA samples were labelled with Cy5 and hybridized against a common reference (5 microg of a mix of all RNA samples used in the experiment) labelled with Cy3 using the ChipShotTM Direct Labeling and Clean-Up system (Promega) according to the manufacturer instructions for total RNA labeling.
|
| |
|
| |
| Hybridization protocol |
Hybridizations were performed in a Discovery XT System hybridization robot using the ChipMap 80 kit (Ventana Medical Systems, Tucson, AZ, USA) at INRA SCRIBE transcriptomic facilities (IFR 140 GFAS, Rennes, France). Prehybridization and hybridization were performed using INRA SCRIBE dedicated protocol. Briefly, prehybridization was performed in 2 ml of ChipSpread buffer containing 4X SSC and 0.2 % SDS (1 h, 42 °C). Before hybridization, target cDNAs labelled with either Cy5 or Cy3 were mixed and hybridized (6h, 42 °C). Microarrays were then washed manually at room temperature in 1X SSC.
|
| Scan protocol |
Images of the microarrays were generated with a Genepix 4000B scanner and Genepix Pro analysis software v6.0 (Axon Instruments, Molecular Devices Co., Sunnyvale, CA, USA).
|
| Description |
Hybridization OK
|
| Data processing |
Analysed with R software and the package Limma (Smyth 2004). Aberrant spots (flag value < -49) were removed. Median values were background corrected with the “normexp” procedure (Ritchie et al. 2007, offset =35) then loess normalized (Colantuoni et al. 2002) then Gquantile normalized (Yang and Thorne 2003). A linear model applied to perform the differential analysis considering animals pairing.
|
| |
|
| Submission date |
Sep 24, 2009 |
| Last update date |
Oct 01, 2009 |
| Contact name |
Isabelle Luron |
| E-mail(s) |
[email protected]
|
| Organization name |
National Institute for Agronomical Research (INRA)
|
| Department |
Human Nutrition Division
|
| Lab |
Nutrition & Digestive, Nervous and Behaviourial Adaptation
|
| Street address |
Domaine de la Prise
|
| City |
Saint-Gilles |
| State/province |
Rennes |
| ZIP/Postal code |
35590 |
| Country |
France |
| |
|
| Platform ID |
GPL7435 |
| Series (1) |
| GSE18249 |
Postnatal intestinal response of IUGR piglets to high proteins before weaning |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Limma normalized log ratio (test/reference); GenePix-flagged values removed |
| CH1_MEDIAN |
channel 1 signal intensity median - Cy5 |
| CH1_MEAN |
channel 1 signal intensity mean |
| CH1_SD |
channel 1 signal intensity standard deviation |
| CH1_BKD_MEDIAN |
channel 1 background signal intensity median |
| CH1_BKD_MEAN |
channel 1 background signal intensity mean |
| CH1_BKD_SD |
channel 1 background signal intensity standard deviation |
| CH2_MEDIAN |
channel 2 signal intensity median - Cy3 |
| CH2_MEAN |
channel 2 signal intensity mean |
| CH2_SD |
channel 2 signal intensity standard deviation |
| CH2_BKD_MEDIAN |
channel 2 background signal intensity median |
| CH2_BKD_MEAN |
channel 2 background signal intensity mean |
| CH2_BKD_SD |
channel 2 background signal intensity standard deviation |
| FLAGS |
|
| UNF_VALUE |
Limma normalized log ratio (test/reference) |
