RESUMEN
Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , ARN/química , Factores de Transcripción SOX/genética , Factores de Transcripción/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Transcriptoma , Proteínas Señalizadoras YAPRESUMEN
In this work, we present a promising diagnostic tool for melanoma diagnosis. With the proposed terahertz biosensor, it was possible to selectively and sensitively detect the early growth response protein 2, a transcription factor with an increased activity in melanoma cells, from a complex sample of cellular proteins. Fundamentally, the sensor belongs to the frequency selective surface type metamaterials and consists of a two-dimensional array of asymmetrically, doubly split ring resonator unit cells. The single elements are slits in a metallic layer and are complemented by an undercut etch. This allows a selective functionalization of the active area of the sensor and increases the sensitivity towards the target analyte. Hereby, specific detection of a defined transcription factor is feasible.
Asunto(s)
Melanoma , Factores de Transcripción , Humanos , Melanoma/diagnóstico , Regulación de la Expresión Génica , Agitación PsicomotoraRESUMEN
Pigmentation is an important process in skin physiology and skin diseases and presumably also plays a role in Parkinson's disease (PD). In PD, alpha-Synuclein (aSyn) has been shown to be involved in the pigmentation of neurons. The presynaptic protein is intensively investigated for its pathological role in PD, but its physiological function remains unknown. We hypothesized that aSyn is both involved in melanocytic differentiation and melanosome trafficking processes. We detected a strong expression of aSyn in human epidermal melanocytes (NHEMs) and observed its regulation in melanocytic differentiation via the microphthalmia-associated transcription factor (MITF), a central regulator of differentiation. Moreover, we investigated its role in pigmentation by performing siRNA experiments but found no effect on the total melanin content. We discovered a localization of aSyn to melanosomes, and further analysis of aSyn knockdown revealed an important role in melanocytic morphology and a reduction in melanosome release. Additionally, we found a reduction of transferred melanosomes in co-culture experiments of melanocytes and keratinocytes but no complete inhibition of melanosome transmission. In summary, this study highlights a novel physiological role of aSyn in melanocytic morphology and its so far unknown function in the pigment secretion in melanocytes.