|
| Status |
Public on May 01, 2006 |
| Title |
Livers from fat line chickens, pool 6, replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Livers from fat line chickens
|
| Organism |
Gallus gallus |
| Characteristics |
Livers from four fat line chickens with 2.3 % (mean) of adipose tissue content per live body weight
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA were extracted with TRIzol Reagent (Invitrogen) from 4 chickens. Poly(A)+ RNA was extracted from a pool of the 4 total RNA preparations with PolyATtract® mRNA isolation system III (Promega)
|
| Label |
Cy5
|
| Label protocol |
Poly(A)+ RNA (500 ng) were reverse-transcribed with amino-allyl-dUTPs (CyScribe Post-Labeling Kit, Amersham Biosciences). The amino-allyl modified cDNAs were purified on GFX columns (GFX Purification Kit, Amersham Biosciences) and further labelled by incubation with cyanine dyes (Cy3 or Cy5). Labeled cDNAs were also purified on GFX columns and mixed with hybridization buffer (5X Denhardt’s, 3.5X SSC, 0.3 % SDS, 0.5 µg/µl yeast RNA, 0.5 µg/µl poly(A) RNA and 50 % formamide)
|
| |
|
| Channel 2 |
| Source name |
Livers from all lean and fat line chickens
|
| Organism |
Gallus gallus |
| Characteristics |
As a reference sample, poly(A)+ RNA preparations from all lean and fat line chickens (10) were mixed in a superpool.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
pooling of poly(A)+ RNA prepared from ten lean and fat line chicken liver total RNA pools
|
| Label |
Cy3
|
| Label protocol |
Poly(A)+ RNA (500 ng) were reverse-transcribed with amino-allyl-dUTPs (CyScribe Post-Labeling Kit, Amersham Biosciences). The amino-allyl modified cDNAs were purified on GFX columns (GFX Purification Kit, Amersham Biosciences) and further labelled by incubation with cyanine dyes (Cy3 or Cy5). Labeled cDNAs were also purified on GFX columns and mixed with hybridization buffer (5X Denhardt’s, 3.5X SSC, 0.3 % SDS, 0.5 µg/µl yeast RNA, 0.5 µg/µl poly(A) RNA and 50 % formamide)
|
| |
|
| |
| Hybridization protocol |
Test sample labelled with Cy5 and reference sample labelled with Cy3 solutions (10 µl) were mixed and incubated 2 min at 100°C, and 30 min at 37°C before hybridization. Hybridizations were performed in hybridization chambers (Corning Life Sciences) overnight at 42°C and slides were washed in 2X SSC, 0.1 % SDS, then in 1X SSC and finally twice in 0.2X SSC.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 with ScanArray 4000 XL (Packard Biochip Technologies) fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 4.0.1.17 analysis software.
|
| Description |
Livers from fat line chickens, pool 6, replicate 2
|
| Data processing |
After background correction and removal of flagged values, log base 2 expression ratios were normalized by lowess fitness using Madtools.
|
| |
|
| Submission date |
Dec 19, 2005 |
| Last update date |
Dec 19, 2005 |
| Contact name |
Christian Diot |
| E-mail(s) |
Christian.Diot@rennes.inra.fr
|
| Organization name |
INRA
|
| Department |
Animal Genetics
|
| Lab |
UMR Génétique Animale
|
| Street address |
65, rue de saint-Brieuc, CS 84215
|
| City |
Rennes cedex |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL3265 |
| Series (1) |
| GSE3867 |
Gene expression in chicken livers from lean and fat lines |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Normalized log2 ratio of median defined by CH1/CH2 |
| CH1_median |
CH1 (F635) median fluorescence intensity |
| CH1_MEAN |
CH1 (F635) mean fluorescence intensity |
| CH1_SD |
CH1 (F635) fluorescence intensity standard deviation |
| CH1_BKD_Median |
CH1 (F635) background median fluorescence intensity |
| CH1_BKD_MEAN |
CH1 (F635) background mean fluorescence intensity |
| CH1_BKD_SD |
CH1 (F635) background fluorescence intensity standard deviation |
| % > CH1_BKD_+2SD |
% of feature pixels greater than two standard deviations over the background |
| CH2_median |
CH2 (F532) median fluorescence intensity |
| CH2_MEAN |
CH2 (F532) mean fluorescence intensity |
| CH2_SD |
CH2 (F532) fluorescence intensity standard deviation |
| CH2_BKD_Median |
CH2 (F532) background median fluorescence intensity |
| CH2_BKD_MEAN |
CH2 (F532) background mean fluorescence intensity |
| CH2_BKD_SD |
CH2 (F532) background fluorescence intensity standard deviation |
| % > CH2_BKD_+2SD |
% of feature pixels greater than two standard deviations over the background |
| Ratio of Medians (635/532) |
Unnormalized ratio of medians defined by CH1/CH2 |
| Ratio of Means (635/532) |
Unnormalized ratio of means defined by CH1/CH2 |
| AREA |
Number of feature pixels |
| BKD_AREA |
Number of feature background pixels |
| CH1_Median - CH1_BKD |
Channel 1 median signal |
| CH2_Median - CH2_BKD |
Channel 2 median signal |
| CH1_Mean - CH1_BKD |
Channel 1 mean signal |
| CH2_Mean - CH2_BKD |
Channel 2 mean signal |
