జర్నల్ ఆఫ్ స్టెమ్ సెల్ రీసెర్చ్ & థెరపీ

నైరూప్య

Laser Capture Microdissection of Murine Interzone Cells: Layer Selection and Prediction of RNA Yield

Florien Jenner, Gerjo JVM van Osch, Mairead Cleary, Iris Ribitsch, Ulrich Sauer, René van Weeren and Pieter Brama

Objective: Articular chondrocytes originate from a distinct cohort of progenitor cells located in the so-called interzone in embryonic developing joints. We conducted this study 1) to determine if it is possible to identify the intermediate and outer interzone layers histologically (using cresyl violet) without adjunct in situ hybridization or immunohistochemistry; 2) to establish whether sufficient amounts of RNA can be harvested from each interzone layer of individual embryos to allow gene expression analysis; 3) to develop measurements that can provide an estimation of the RNA yield prior to costly amplification steps. Methods: Cells from the outer (OI) and intermediate (II) interzone of the nascent femorotibial joint and the epiphyseal cartilage (EC) of femur and tibia of murine embryos of 13.5 and 15.5 days gestation were harvested using laser capture microdissection (LCM). Subsequently, microarray analysis was performed to confirm appropriate layer selection. The surface area harvested and the grey value (gv) of photomicrographs taken during LCM was measured and the corresponding relative optical density (ROD) was calculated and degree and significance of the correlation with RNA yield were determined. Results: Cells from the OI, II and EC could be identified histologically using cresyl violet staining and were successfully harvested with LCM yielding sufficient amounts of RNA for linear amplification and microarray analysis. The RNA yield correlated significantly with the tissue surface area harvested, the mean gv and the corresponding ROD. Conclusions: This study provides a technique for selective laser capture microdissection and subsequent microarray analysis of murine interzone cells of the intermediate and outer layer and presents a method to estimate the RNA yield by measuring the tissue area harvested and calculating the ROD. We recommend to harvest a minimum of 1×106 μm2 of 13.5 days murine embryos and 3×106 μm2 of 15.5 days murine embryos to obtain approximately 10 ng total RNA that can be used for linear T7-based amplification and subsequent microarray analysis.