Hypoxia response

The goal of the study was to identify hypoxia-induced gene expression changes in C. elegans and to determine which of these responses to hypoxia were regulated by hif-1. Towards these aims, we analyzed mRNA expression patterns in synchronized populations of wild-type worms that were cultured in standard lab conditions (normoxia) or in hypoxia. We also assayed mRNA from two mutant strains: (i) animals carrying the strong loss-of-function mutation in hif-1 and (ii) C. elegans that carry a deletion in vhl-1 and express the HIF-1 protein at constitutively high levels. In the microarray experiments, 3 strains were assayed: wild type N2, hif-1 (ia04), and vhl-1 (ok161). Worms were incubated for 4 hours in 21% oxygen or 0.1% oxygen at 210C. Animals were quickly harvested in ice cold M9 buffer, and poly (A) RNA was isolated using established procedures. No more than 3 minutes elapsed between the removal of plates from the hypoxic chamber and the addition of Trizol. RNA was isolated from three independent experiments for each experimental condition (3 genotypes; normoxia vs hypoxia). mRNAs were hybridized to whole-genome microarrays which contained probes for 17,817 predicted genes (94% of the genome). Ch1 in the array represents the data for the worms growed under hypoxia; and Ch2 in the array reprents the data for the worms growed under normoxia. Keywords: Biological Replicates本研究旨在鉴定秀丽隐杆线虫（C. elegans）中缺氧诱导的基因表达变化，并明确其中哪些缺氧应答过程受hif-1调控。基于上述研究目标，我们对同步化的野生型线虫种群的mRNA表达模式进行了分析，这些线虫分别在标准实验室培养条件（常氧（normoxia））或缺氧环境中培养。我们还对两种突变株系的mRNA进行了检测：（i）携带hif-1强功能丧失突变的线虫，（ii）vhl-1基因缺失且组成型高表达HIF-1蛋白的秀丽隐杆线虫（C. elegans）。在本次微阵列（microarray）实验中，共检测了3个株系：野生型N2、hif-1(ia04)突变株以及vhl-1(ok161)突变株。线虫分别在21%氧浓度、0.1%氧浓度的环境中于21℃孵育4小时。迅速将线虫用冰预冷的M9缓冲液收集，并通过已建立的实验流程分离聚腺苷酸（poly(A)）RNA。从缺氧培养箱取出培养板至加入Trizol试剂的间隔时间不超过3分钟。针对每种实验条件（3种基因型；常氧与缺氧对照），我们均通过3次独立实验完成RNA提取。将mRNA与全基因组微阵列进行杂交，该微阵列包含针对17817个预测基因的探针（覆盖该基因组的94%）。该微阵列的Ch1通道对应缺氧环境下培养的线虫的检测数据，Ch2通道对应常氧环境下培养的线虫的检测数据。关键词：生物学重复（Biological Replicates）