These values are in line with the reported quality control requirements. The 3′/m ratio means of GAPDH and β-Actin ranged between 1.11–1.56 and 1.41–2.12, respectively. Under the empirically set LCM parameters, four homogeneous cell populations were efficiently isolated from their respective zones.
The improved morphology of the frozen sections of our protocol has extended the range of cell types to be isolated. Both fixed and unfixed tissue sections allowed reliable identification of MCC zones. Then, to evaluate LCM-RNA integrity, 3′/m ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin ( β-Actin) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were calculated. Additional specimens were microdissected to prepare LCM samples from FCC and each MCC zone individually. Formalin-fixed and frozen unfixed tissue sections were prepared and compared histologically. MCC and FCC (femoral condylar cartilage) specimens were harvested from 5-week-old Sprague–Dawley male rats. Therefore, our aim is to optimize an LCM protocol for isolating four homogenous zone-specific cell populations from their respective MCC zones while preserving the quality of RNA recovered. Laser capture microdissection (LCM) technology allows isolating zonal (homogenous) cell populations and consequently generating more accurate molecular and genetic data, but the challenges during tissue preparation and microdissection procedures are to obtain acceptable tissue section morphology that allows histological identification of the desirable cell type and to minimize RNA degradation.
Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis. Mandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones.
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