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Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection.

ABSTRACT: To characterize the gene expression profile of regenerated melanocytes in the narrow band UVB (NBUVB)-treated vitiligo epidermis and their precursors in the hair follicle, we present here a strategy of RNA isolation from in situ melanocytes using human frozen skin. We developed a rapid immunostaining protocol using the NKI-beteb antibody, which labels differentiated and precursor melanocytes, followed by fluorescent laser capture microdissection. This technique enabled the direct isolation, from melanocyte and adjacent keratinocyte populations, of satisfactory quality RNA that was successfully amplified and analysed by qRT-PCR. The melanocyte-specific gene transcripts TYR, DCT, TYRP1 and PMEL were significantly upregulated in our NBUVB-treated melanocyte samples as compared with the keratinocyte samples, while keratinocyte-specific genes (KRT5 and KRT14) were expressed significantly higher in the keratinocyte samples as compared with the melanocyte samples. Furthermore, in both NBUVB-treated vitiligo skin and normal skin, when bulge melanocytes were compared with epidermal melanocytes, we found significantly lower expression of melanocyte-specific genes and significantly higher expression of three melanocytic stem cell genes (SOX9, WIF1 and SFRP1), while ALCAM and ALDH1A1 transcripts did not show significant variation. We found significantly higher expression of melanocyte-specific genes in the epidermis of NBUVB-treated vitiligo, as compared to the normal skin. When comparing bulge melanocyte samples from untreated vitiligo, NBUVB-treated vitiligo and normal skin, we did not find significant differences in the expression of melanocyte-specific genes or melanocytic stem cell genes. These techniques offer valuable opportunities to study melanocytes and their precursors in vitiligo and other pigmentation disorders.

SUBMITTER: Goldstein NB

PROVIDER: S-EPMC5322465 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection.

Goldstein Nathaniel B NB Koster Maranke I MI Hoaglin Laura G LG Wright Michael J MJ Robinson Steven E SE Robinson William A WA Roop Dennis R DR Norris David A DA Birlea Stanca A SA

Experimental dermatology 20161001 10

To characterize the gene expression profile of regenerated melanocytes in the narrow band UVB (NBUVB)-treated vitiligo epidermis and their precursors in the hair follicle, we present here a strategy of RNA isolation from in situ melanocytes using human frozen skin. We developed a rapid immunostaining protocol using the NKI-beteb antibody, which labels differentiated and precursor melanocytes, followed by fluorescent laser capture microdissection. This technique enabled the direct isolation, from ...[more]

PMID: 27193292

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Project description:Mandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones. Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis. 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. 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. MCC and FCC (femoral condylar cartilage) specimens were harvested from 5-week-old Sprague-Dawley male rats. Formalin-fixed and frozen unfixed tissue sections were prepared and compared histologically. Additional specimens were microdissected to prepare LCM samples from FCC and each MCC zone individually. 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. Both fixed and unfixed tissue sections allowed reliable identification of MCC zones. The improved morphology of the frozen sections of our protocol has extended the range of cell types to be isolated. Under the empirically set LCM parameters, four homogeneous cell populations were efficiently isolated from their respective zones. The 3'/m ratio means of GAPDH and β-Actin ranged between 1.11-1.56 and 1.41-2.12, respectively. These values are in line with the reported quality control requirements. The present study shows that the optimized LCM protocol could allow isolation of four homogenous zone-specific cell populations from MCC, meanwhile preserving RNA integrity to meet the high quality requirements for subsequent molecular analyses. Thereby, accurate molecular and genetic data could be generated.

Dataset Information

Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection.

Publications

Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection.

Similar Datasets

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