Figure 4. Laser capture microdissection of a human growth plate and QRT-PCR of COL27A1.
An eosin-stained frozen section of growth plate cartilage from a 7-month-old human phalanx demonstrated laser capture of hypertrophic zone chondrocytes. The tissue is shown before (A) and after (B) laser activation melted polymer around the cells of interest. The microdissected hypertrophic cells attached to the polymer film substrate are shown in panel C. Bar = 100 μm for panels A–C. Captured cells were used to obtain Sybr Gold-stained gels and QRT-PCR of COL27A1 and 18S rRNA (D). Reaction buffers without cDNA served as a negative control. RNA isolated from human articular chondrocytes grown in vitro was used as a positive control. Normalized to 18S rRNA, levels of COL27A1 were 16-fold greater [difference of 4 threshold cycles (Cts)] in resting/proliferative cells than in hypertrophic chondrocytes.