INRA/UMR Biologie du Développement et Reproduction
Treatment protocol
Tissues were collected and immediately snap-frozen.
Extracted molecule
total RNA
Extraction protocol
RNA samples: Corpus luteum (n=8, a day per diet) - Oviduct (n=8, a day per diet. Pooled RNA from ipsi-contra biopsies) - Endometrium (n=8, a day per diet. Pooled RNA from ipsi-contra biopsies). Total RNA from endometrium, corpus luteum and oviduct were extracted with Chomczynski & N. Sacchi protocol and purified using Rneasy Mini kit (Quiagen). RNA quality was first verified by A260/280 absorbance ratios.
Label
P33
Label protocol
30µg of total RNA were retro-transcribed and directly labelled with 33P dATP as described (Degrelle et al., 2008). Total RNA were mixed with 500ng of random hexamers in a volume of 25µl. The mixture was incubated at 70°C for 10 min and chilled on ice. cDNA was synthesised by the addition of 5µl 10X PCR buffer, 5µl 25mM MgCl2, 5 µl 0,1 mM DTT, 2,5µl 10mM mix dGTP, dCTP and dTTP, 2,5µl water, 50 µCi 33P dATP and 200U Superscript II (Invitrogen) at 42°C for 50min. The RNA template was removed by the addition of 1µl RNAse H- and incubation at 37°C for 20 min. Labelled targets were then purified on Sephadex columns (G-50).
Hybridization protocol
Prehybridisation in ExpressHybTM Hybridization Solution (Clontech) at 68°C during 1h (14ml). Hybridisation using new ExpressHybTM Hybridization Solution (Clontech) at 68°C overnight (10ml). Arrays were washed four times in 2X SSC, 1% SDS and once in 0.1X SSC, 0.5% SDS at 68°C for 30 min each. They were then exposed to phosphor-screens (Amersham) for 5 days as described in Degrelle et al., 2008.
Scan protocol
Scanner: STORM 760 from Molecular Dynamics. Raw data set: Feature extraction with Imagene 5.5 software from BioDiscovery (Proteigene)
Description
Syntax of the sample names: Tissue_Diet_Individual_DayOfCycle. Where the tissue is either the oviduct, the endometrium or the corpus luteum. Where the diet is control or underfed. Where the day of cycle is either D4, D8, D12, D15. Experimental variables: Diet = control (100% INRA requirements during 140 days), underfed (40% of control diet during the dry period (60 days), 80% of control diet during the first 80 days of postpartum). Additional data: - High producing dairy cows (n=8). Holstein breed. Induced ovulation in post-partum - Surgical tissue sampling at 4 days (a day per diet, n=8) - Corpus Luteum: biopsies at one day per diet (n=8) - Oviduct: ipsi- and contra-lateral biopsies per day, and diet (n=16) - Endometrium: ipsi- and contra-lateral biopsies per day, and diet (n=16)
Data processing
Intensity = Signal Mean. Normalization = Signal mean were log2 transformed and median centred. To normalize our data, we used R version 2.13.0. As the data were only log2 transformed and median centered (=basic R functions), we didn't used any microarray packages.
