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

Goldstein, Nathaniel B.; Koster, Maranke I.; Hoaglin, Laura G.; Wright, Michael; Robinson, Steven E.; Robinson, William A.; Roop, Dennis R.; Norris, David A.; Birlea, Stanca A.

doi:10.1111/exd.13072

Cited by 9 publications

(12 citation statements)

References 25 publications

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“…Device application in accordance with our protocol is essential for obtaining reliable results as shown by the difference in RNA amount recovered with different application protocols (Figure 3). Once the sample is extracted, the subsequent sample processing step for RNA extraction is highly similar to established protocols 15,16 . Besides from the initial steps in RNA extraction that are modified for the absorbent microbiopsy, another key change is the use of RNase-free water to improve the results for downstream applications.…”

Section: Discussionmentioning

confidence: 99%

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Lei

Yamada

Hoang

et al. 2019

JoVE

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Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

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Section: Discussionmentioning

confidence: 99%

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Lei

Yamada

Hoang

et al. 2019

JoVE

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“…In this study, we report an application (which, to our knowledge, is previously unreported), consisting of rapid immunostaining combined with F-LCM [to isolate RNA from specific cells (melanocytes), located at specific sites (bulge and epidermis)], and with RNA sequencing. In a previous study, we showed that the RNA captured from the HF bulge and epidermal melanocytes of the NBUVB-treated vitiligo and normal skin is of satisfactory quality for qRT-PCR analysis (Goldstein et al, 2016). Our current application offers a better characterization of melanocyte populations in the regenerated epidermis and the bulge of the NBUVB-treated vitiligo skin, because it uses RNA-Seq technology that has deeper coverage and higher sensitivity than qRT-PCR.…”

Section: Discussionmentioning

confidence: 99%

“…epidermal melanocytes. Using immunostaining and qRT-PCR, we previously identified TYR upregulation in the epidermal melanocytes of NBUVB-treated vitiligo skin and unremarkable TYR expression in the bulge melanocyte precursors (Goldstein et al, 2015; 2016). Indeed, our RNA-seq study confirmed the low expression of TYR in the NBUVB-treated vitiligo bulge melanocytes (Fig.…”

Section: Discussionmentioning

confidence: 99%

Repigmentation of Human Vitiligo Skin by NBUVB Is Controlled by Transcription of GLI1 and Activation of the β-Catenin Pathway in the Hair Follicle Bulge Stem Cells

Goldstein

Koster

Jones

et al. 2018

Journal of Investigative Dermatology

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Vitiligo repigmentation is a complex process in which the melanocyte-depleted interfollicular epidermis is repopulated by melanocyte precursors from hair follicle bulge that proliferate, migrate, and differentiate into mature melanocytes on their way to the epidermis. The strongest stimulus for vitiligo repigmentation is narrow-band UVB (NBUVB), but how the hair follicle melanocyte precursors are activated by UV light has not been extensively studied. To better understand this process, we developed an application that combined laser capture microdissection and subsequent whole transcriptome RNA sequencing of hair follicle bulge melanocyte precursors and compared their gene signatures to that of regenerated mature epidermal melanocytes from NBUVB-treated vitiligo skin. Using this strategy, we found up-regulation of TNC, GJB6, and THBS1 in the hair follicle bulge melanocytes and of TYR in the epidermal melanocytes of the NBUVB-treated vitiligo skin. We validated these results by quantitative real-time-PCR using NBUVB-treated vitiligo skin and untreated normal skin. We also identified that GLI1, a candidate stem cell-associated gene, is significantly up-regulated in the melanocytes captured from NBUVB-treated vitiligo bulge compared with untreated vitiligo bulge. These signals are potential key players in the activation of bulge melanocyte precursors during vitiligo repigmentation.

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“…Furthermore, we detected a robust decrease in the transcript level of MITF(M), the master regulator of the melanocyte differentiation program, upon Rec KD in A375 cells ( Figure 4B). In addition to MITF, we also observed a decreased expression of other melanocyte differentiation markers (e.g., TYR, PMEL) [90] ( Figure 4C). Together, the transcriptome changes we observed upon Rec KD in A375 cells suggests that Rec expression is part of a regulatory cascade inhibiting the EMT-like process.…”

Section: Depleting Herv-k-rec May Induce An Emt-like Process In A375 mentioning

confidence: 82%

Human Endogenous Retrovirus K Rec forms a regulatory loop with MITF that opposes the progression of melanoma to an invasive stage

Singh

Cai

Bunse

et al. 2020

Preprint

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In the human genome, HERV-K(HML2) is the most recently endogenized retrovirus 42 Through its interaction with CRM1 nuclear export factor [4, 6], Rec is involved in exporting un-43 spliced HERV-K RNA from the nucleus [3, 7]. In addition, Rec can directly modulate cell signalling 44 via binding to the promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor of 45 the c-MYC proto-oncogene [8]. Rec also binds the TZFP (testicular zinc-finger protein) and the hSGT 46 2 of 23 (human small glutamine-rich tetratricopeptide repeat protein), which are transcriptional repressors 47 of the androgen receptor [9, 10]. While it is evident that Rec must have first evolved to facilitate 48 HERV-K replication, there is speculation that the protein may have been coopted for physiological 49 function, such as spermatogenesis [6, 11] and defence against viral infections in the early human 50 embryo [12]. 51It has been reported that at least 18 HERV-K genomic loci can be transcribed in healthy human 52 tissues and express potentially-coding Rec or Np9 mRNAs (alone or in combination with retroviral 53 genes), though the landscape of transcribed HERV-K loci differs considerably between cell/tissue 54 types [13]. Rec RNA/protein expression has been observed in human embryonic tissues, placenta and 55 retina [14, 15]. Interestingly, HERV-K is expressed in both germinal-and pluripotent stem cells, 56 providing a special link between these cell types [16]. While HERV-K transcription has been observed 57 at low levels in various normal tissues, it is strongly induced upon environmental stress, such as 58 ultraviolet irradiation (e.g. UVB and UVC), starvation or viral infection that may manifest the global 59 DNA hypomethylation [17][18][19][20][21]. Furthermore, many studies have reported robust mRNA 60 upregulation of several genomic loci of HERV-K in various disease states, including certain 61 autoimmune diseases and a number of cancers, especially lung, breast cancers, germ cell tumours 62 and melanoma [18, 22, 23] and reviewed in [24, 25]. Moreover, transcriptional activation and 63 translation of various HERV-K products [22, 26] and even virus-like particles have been observed in 64 certain cancer cells such as teratoma, embryonic carcinoma and melanomas [3,[27][28][29]. 65Whether and how the overexpression of HERV-K products may contribute to the etiology or 66 progression of these diseases has been the subject of many studies, debates, and speculations. Several 67 mechanisms have been proposed by which HERV-K might contribute to disease. The activated LTRs 68 could act as alternative promoters and de-regulate tumor suppressor genes or proto-oncogenes 69 (reviewed in [35, 36]). Furthermore, the Env protein of HERV-K, via its fusogenic property, is capable 70 to induce cell-cell fusion and could therefore contribute to tumour invasiveness [18, 37, 38]. Env could 71 also have oncogenic properties through direct interference with cellular signalling pathways (e.g. 72RSS/MEK/ERK) [39, 40]. Indeed, the overexpressi...

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

Cited by 9 publications

References 25 publications

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Repigmentation of Human Vitiligo Skin by NBUVB Is Controlled by Transcription of GLI1 and Activation of the β-Catenin Pathway in the Hair Follicle Bulge Stem Cells

Human Endogenous Retrovirus K Rec forms a regulatory loop with MITF that opposes the progression of melanoma to an invasive stage

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