Background

Circadian clock is found all over the body and in the lung it is shown to strongly oscillate in Club cells (Gibbs et al., 2009 and 2014). Whole lung circadian gene profiling is carried before in mice and rat (Sukumaran et al., 2011; Zhang et al., 2014). However, at least 15% of those circadian genes are coming from leukocytes (Haspel et al., 2014). It is useful but difficult to get cell type specific circadian gene profiling, given the high heterogeneity of cell population in tissues. Tissue digestion followed by flow cytometry sorting is usually to solve this problem but it is not clearly whether gene expression will change during this process. An alternative way is to use laser capture microdissection to take target cells from fixed tissues, Note that this could lead to comprised cell population purity. Specific cell population can be identified by antibody staining or anatomic information. In terms of Club cells in lung, they are known to be enriched in distal bronchiolar epithelium. A protocol showing how to do microarray using RNA from Club cells by laser capture (PixCell II Laser Capture Microdissection system) was published more than 10 years before and there are some advances in the technique (Betsuyaku and Senior, 2004; Cummings et al., 2011). Here I present a detailed protocol on circadian gene profiling in Club cell RNA by laser capture microdissection in Leica LMD6500 system. It covers from mouse tissue collection to get RNA samples ready for RNA seq library construction. Generally, to get good quality RNA, all tools and working areas should keep RNase contamination to the minimum (like using RNaseZAP solution to clean and keep sections dry in microdissection). For all the downstream steps, please refer to the published protocol by Jiajia Li, etc. (Li et al., 2015).