Total RNA extracted from a pool of 15 to 20 mm antral follicles, determined as healthy using microscopic examination after Feulgen staining of some granulosa cells, originating from cow 9936
annotation pf/mf/gf: GF group: large follicles date hybridation-genovul: 2008-03-25 status: S (healthy follicles) animal-number_genovul: 9936
Extracted molecule
total RNA
Extraction protocol
Once ovaries collected, all visible antral follicles larger than 1 mm in diameter for sows and 3 mm for cows were isolated carefully using a binocular microscope. After dissection, follicle diameter was measured and each follicle was classified according to size, animal and follicular quality. Follicles were allocated to two size classes: small (noted SF, 1-2 mm for sows and 3-5mm for cows) and large (noted LF, 7-8 mm for sows and 15-20mm for cows). Granulosa cells were recovered from all individual follicles and they were stored at -80°C until RNA extraction. For each follicle, a sample of granulosa cells was smeared on a histological slide, and then Feulgen stained to determine follicular quality by microscopic examination. Only healthy follicles (presence of mitosis and absence of pycnosis in granulosa cells) were kept for this study. RNA was extracted from granulosa cells according to the technique described by Chomczynski and Sacchi with minor modifications using pools of granulosa cells from the same follicle size class and the same animal. The quality of each RNA sample was checked through the Bioanalyser Agilent 2100 (Agilent Technologies, Massy, France) and low-quality RNA preparations were discarded.
Label
P33
Label protocol
The arrays were hybridized with 33P-labelled complex probes synthesized from 5 µg of each RNA sample, using SuperScript II RNAse H-reverse transcriptase (Invitrogen, Cergy-Pontoise, France)
Hybridization protocol
Micro-arrays were first hybridized with a 33P-labelled oligonucleotide sequence present in all PCR products to control the quality of the spotting process and to evaluate the amount of DNA in each spot. After stripping, the arrays were hybridized with 33P-labelled complex probes synthesized from 5 µg of each RNA sample, using SuperScript II RNAse H-reverse transcriptase (Invitrogen, Cergy-Pontoise, France). Each complex probe has been hybridized on membrane exposed 1, 3 or 7 days to radioisotopic-sensitive imaging plates (BAS-2025, Fujifilm, Raytest, Courbevoie, France).
Scan protocol
The imaging plates were scanned thereafter with a phosphor imaging system at 25µm resolution (BAS-5000, Fujifilm, Raytest, Courbevoie, France). Hybridization images obtained from oligonucleotide and complex probes were quantified using the semi-automated AGScan software.
Description
A pig nylon micro-array is hybridized with a complex 33P labelled probe
Data processing
Data exploration and analysis were performed using R software. The data coming from complex probes hybridization were analysed on the logarithmic scale. As a first step, negative spot with >7 intensity values were checked on images and replaced by the median of negative spots in case of obvious overshining effect or hybridization stain. Then, luciferase positive controls were removed and the correlations of the data from each membrane were examined intra condition. Hybridizations with a lower than 0.80 correlation coefficient were discarded. Spots with low signal value (below the average of empty spots + 3 standard deviations) were considered as unexpressed and were excluded from the analysis. Finally, the negative control spots were removed and the remaining data were centred for each membrane.
