In vivo gene expression in granulosa cells during pig terminal follicular development.. Sus scrofa

The aim of this study is the identification of genes and gene networks involved in pig ovarian follicular development. cDNA nylon micro-arrays (2849 sequenced clones) were designed from different libraries : four subtractive suppressive hybridization libraries (generated from small versus large and small versus medium follicles) and a pig multi-tissue cDNA library. Granulosa cells were isolated from healthy follicles (small, medium or large), 24 h or 96 h after the end of a progestagen treatment. The RNA isolated from these cells was used to hybridize the micro-arrays and hybridisations were performed in duplicate. The images were quantified using Bzscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Keywords: cell type comparison Overall design: Granulosa cells were isolated from healthy follicles (small, medium or large). The RNA isolated from these cells was used to hybridize nylon micro-arrays. The arrays contained PCR products from 2848 pig cDNA clones (GPL3978), spotted in duplicate on two separate fields of the same membrane (last number 1 of the local ID corresponds to the left field and 2 corresponds to right field). The data have been generated from 14 RNA follicle pools (five Large, five Medium and four Small follicles pools). For each follicle pool, 2 radioactive labellings were performed (before last number of the local ID corresponds to the duplicate number). Each membrane was exposed 6, 12 or 24 hours (depending on the saturation signal) to radioisotopic-sensitive imaging plates (BAS-2025, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The imaging plates were scanned thereafter with a phosphor imaging system at a 25 µm resolution (BAS-5000, Fujifilm, Raytest France S.A.R.L., Courbevoie, France). The images were quantified using semi-automated software, BZScan (Lopez F, Rougemont J, Loriod B, Bourgeois A, Loi L, Bertucci F, Hingamp P, Houlgatte R, and Granjeaud S. Feature extraction and signal processing for nylon DNA microarrays. BMC Genomics 5: 38, 2004). Fixed circle segmentation, i.e. a grid process with a fixed spot diameter was applied. The hybridization images were quantified by the extraction of the intensity for each spot. A new version of BZsan (AGscan solfware) is available at : http://mulcyber.toulouse.inra.fr/projects/agscan/ The experimental data were managed using the free BASE software (Saal LH, Troein C, Vallon-Christersson J, Gruvberger S, Borg A, and Peterson C. BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data. Genome Biol 3: SOFTWARE0003, 2002.) modified by Sigenae team to accept radioactive experiments. Finally, data consisted in (14 RNA x 2 labellings x 2 fields) 14 probes, 28 hybridisations, 42 images (14 images were filtred out for background too hight).

本研究旨在鉴定参与猪卵巢卵泡发育的基因及基因调控网络。实验所用的尼龙膜cDNA微阵列（cDNA nylon micro-arrays）包含2849个已测序克隆，其构建自四类文库：四个抑制性消减杂交文库（subtractive suppressive hybridization library，分别由小卵泡与大卵泡、小卵泡与中等卵泡构建）以及一个猪多组织cDNA文库。研究人员从健康卵泡（分为小、中、大三类）中分离颗粒细胞，采集时间为孕激素处理结束后24小时或96小时。提取上述细胞的总RNA用于微阵列杂交实验，每组实验均设置重复杂交。图像量化采用BZScan软件，数据管理采用BASE软件，统计分析使用R软件。

关键词：细胞类型比较
整体实验设计：从健康卵泡（小、中、大三类）中分离颗粒细胞，提取总RNA用于尼龙膜cDNA微阵列杂交。该微阵列包含2848个猪cDNA克隆的PCR扩增产物（GPL3978），同一张膜的两个独立点样区域均设置了重复探针（本地ID的最后一位数字1对应左侧区域，2对应右侧区域）。

本实验数据来自14个卵泡RNA混合样本：其中大卵泡样本5个、中等卵泡样本5个、小卵泡样本4个。每个卵泡混合样本均进行2次放射性标记（本地ID中末位前的数字代表重复实验编号）。每张杂交膜分别暴露于放射性敏感成像板（BAS-2025，富士胶片，Raytest法国SARL公司，库尔伯瓦，法国）6、12或24小时，曝光时长根据信号饱和度调整。随后使用磷光成像系统以25μm分辨率扫描成像板（BAS-5000，富士胶片，Raytest法国SARL公司，库尔伯瓦，法国）。

图像量化采用半自动软件BZScan（引用文献：Lopez F, Rougemont J, Loriod B, Bourgeois A, Loi L, Bertucci F, Hingamp P, Houlgatte R, and Granjeaud S. Feature extraction and signal processing for nylon DNA microarrays. "BMC Genomics" 5: 38, 2004），该方法采用固定圆形分割算法，即基于固定斑点直径的网格定位流程，通过提取每个斑点的信号强度完成杂交图像的量化。BZScan的新版本AGscan软件可在以下网址获取：http://mulcyber.toulouse.inra.fr/projects/agscan/

实验数据采用经Sigenae团队改造的开源BASE软件（BioArray Software Environment，引用文献：Saal LH, Troein C, Vallon-Christersson J, Gruvberger S, Borg A, and Peterson C. BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data. "Genome Biol" 3: SOFTWARE0003, 2002）进行管理，改造后的软件支持放射性杂交实验的数据处理。

最终数据集包含（14组RNA样本 × 2次标记 × 2个点样区域）的实验信息：共14组探针、28次杂交实验、42张图像（其中14张因背景信号过高被过滤排除）。