Gene expression based molecular test as diagnostic aid for the differential diagnosis of psoriasis and eczema in formalin fixed and paraffin embedded tissue, microbiopsies and tape strips
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ABSTRACT:
PROVIDER: PRJNA928395 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23244900_1.fastq.gz | Fastqsanger.gz | |||
| SRR23244900_2.fastq.gz | Fastqsanger.gz | |||
| SRR23245000_1.fastq.gz | Fastqsanger.gz | |||
| SRR23245000_2.fastq.gz | Fastqsanger.gz | |||
| SRR23244901_1.fastq.gz | Fastqsanger.gz |
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Project description:Development of a real-time PCR based molecular classifier to distinguish psoriasis from eczema in FFPE-fixed skin samples and evaluation of the use of minimally invasive microbiopsies and tape strips for molecular diagnosis.
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Project description:Background: Skin biopsies represent a gold standard in skin immunology and pathology but can cause pain and induce scarring. Non-invasive techniques will facilitate study recruitment of e.g. patients with pediatric atopic dermatitis (AD), hand eczema or facial dermatitis. Objective: By RNA sequencing, we examined whether the stratum corneum transcriptome in AD skin can be assessed by tape stripping, as compared to the epidermal transcriptome of AD in skin biopsies. To make the procedure clinically relevant tape strips were stored and shipped at room temperature for up to 3 days. Methods: Nine adult Caucasian AD patients and three healthy volunteers were included. Tape samples were collected from non-lesional and lesional skin. Biopsies were collected from lesional skin and were split into epidermis and dermis. Total RNA was extracted, and shotgun sequencing was performed. Results: Shotgun sequencing could be performed on skin cells obtained from two consecutive tape strips which had been stored and shipped at room temperature for up to three days. The most prominent differences between the tape strip and biopsy derived transcriptome were due to structural genes, while established molecular markers of AD, including CCL17, CCL22, IL17A and S100A7-S100A9, were also identified in tape strip samples. Furthermore, the tape strip derived transcriptome showed promise in also analysing the skin microbiome. Conclusion: Our study shows that the stratum corneum (SC) transcriptome of AD can be assessed by tape stripping the skin, supporting that this method may be central in future skin biomarker research.
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Project description:Hand eczema (HE) is a prevalent skin disease. However, classification of HE into different subtypes remains challenging. Limited number of prior studies have employed invasive biopsy-based strategies; yet, studies of the HE proteome using non-invasive tape stripping methodology have not been reported. In this study, we wanted to assess whether global proteomic analysis of skin tape strip samples can be used for sub-classification of HE patients. Tape strips were collected from patients with HE and healthy skin. Liquid chromatography–mass spectrometry (LC/MS) proteomics was performed, and the global protein expression was analyzed. We identified 2,919 proteins in stratum corneum-derived skin cells from tape strip samples. Compared to healthy skin, the lesional samples from HE patients exhibited increased expression of immune-related markers and a decreased expression of structural barrier proteins. The difference between HE subtypes was restricted to the lesional skin areas, and included an increased expression of skin barrier-related proteins independently of the concurrent AD. In conclusion we found, that the non-invasive tape strip method used in combination with LC/MS proteomics can be used for analysis of skin protein expression in HE patients. Thus, the method shows potential for assessing the proteomic differences between subtypes of HE, and biomarker discovery.
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Project description:Archived formalin-fixed paraffin-embedded (FFPE) samples are the global standard format for preservation of the majority of biopsies in both basic research and translational cancer studies, and profiling chromatin accessibility in the archived FFPE tissues is fundamental to understanding gene regulation. Accurate mapping of chromatin accessibility from FFPE specimens is challenging because of the high degree of DNA damage. Here, we first showed that standard ATAC-seq can be applied to purified FFPE nuclei but yields lower library complexity and a smaller proportion of long DNA fragments. We then present FFPE-ATAC, the first highly sensitive method for decoding chromatin accessibility in FFPE tissues that combines Tn5-mediated transposition and T7 in vitro transcription. The FFPE-ATAC generates high-quality chromatin accessibility profiles with 500 nuclei from a single FFPE tissue section, enables the dissection of chromatin profiles from the regions of interest with the aid of hematoxylin and eosin (H&E) staining, and reveals disease-associated chromatin regulation from the human colorectal cancer FFPE tissue archived for >10 yr. In summary, the approach allows decoding of the chromatin states that regulate gene expression in archival FFPE tissues, thereby permitting investigators to better understand epigenetic regulation in cancer and precision medicine.
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Project description:BackgroundMolecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge.ResultsWe present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97.ConclusionWe developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.
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