Transcription profiling of mouse secondary alveolar septae of neonatal lung tissue

The tips of secondary alveolar septae in day 6 neonatal mouse lung tissue were isolated using laser capture microscopy. RNA was isolated from pooled secondary alveolar tips and also from pooled neonatal day 6 whole lung tissue. The isolated RNAs were then amplified in parallel. Gene array profiling of the two RNA samples was performed. Gene expression in the secondary alveolar septal tips was compared to gene expression in the whole lung tissue. In this way, the genes that are expressed in the tip of secondary alveolar septae were identified as well as the genes that are enriched in the alveolar septal tips versus in whole lung tissue. Experiment Overall Design: We performed an experiment in which we used laser capture microscopy to collect 10,000 secondary alveolar tips from frozen sections of lung tissue obtained from many different day 6 neonatal mice obtained from different litters. In the same experiment, we also isolated RNA from pooled day 6 neonate whole lung tissue. The isolated RNAs were amplified in parallel and then hybridized to microarrays in parallel in order to profile and compare gene expression in the two samples. The entire experiment was performed twice with tissue from different litters. In addition, the amplified RNAs were hybridized to two different microarrays, at different times.本研究采用激光捕获显微切割（Laser Capture Microscopy）技术，从出生6天的新生小鼠肺组织中分离得到次级肺泡隔尖端（secondary alveolar septae tips）。随后，从收集的混合次级肺泡隔尖端样本以及同期收集的混合新生6天全肺组织样本中分别提取RNA，并将两份提取的RNA进行平行扩增。对两份RNA样本开展基因芯片表达谱（gene array profiling）分析，比较次级肺泡隔尖端与全肺组织的基因表达差异，由此鉴定出次级肺泡隔尖端特异性表达的基因，以及相较于全肺组织在次级肺泡隔尖端中富集的基因。 实验整体设计：本实验通过激光捕获显微切割技术，从多窝不同出生6天的新生小鼠的冰冻肺组织切片中收集了10000个次级肺泡隔尖端。同期，从混合的新生6天小鼠全肺组织中提取RNA。将两份提取的RNA进行平行扩增后，同步进行微阵列芯片（microarray）杂交以完成基因表达谱分析并比较二者的表达差异。本实验使用不同窝次的组织重复完成了两次。此外，扩增后的RNA还在不同时间点分别与两款不同的微阵列芯片进行了杂交。