Browsing by Subject "Laser capture microdissection"

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    Optimization of RNA extraction from laser captured microdissected glomeruli from formalin-fixed paraffin-embedded mouse kidney samples for Nanostring analysis.
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2020) Hay, Abigail; Lapointe, Jean-Martin; Lewis, Arthur; Moreno Quinn, Carol; Miranda, Elena
    Optimized protocols for the microdissection of specific areas from archival tissues and the subsequent RNA analysis are needed but challenging due to RNA degradation and chemical modifications. The aim of this study was to present the most appropriate protocol for utilizing mouse FFPE kidney for laser capture microdissection and Nanostring gene expression analysis. We evaluated different section thicknesses (3, 5, 10 μm), 2 RNA extraction kits (Qiagen and Roche) and different H&E staining methods to optimize microdissection and RNA extraction from glomeruli and cortical tubules samples from FFPE mouse kidney. RNA quality and quantity were assessed via Nanodrop and Qubit. The protocol providing the best results consisted of 5 μm sections, a shorter protocol for H&E staining, and RNA extracted with the Roche kit. Higher Nanostring gene counts and lower qPCR cT significantly correlated with RNA concentrations measured with the Qubit, but not with measures obtained with the Nanodrop. The Nanostring data showed that none of the genes included in the panel was differentially expressed in the cortical tubule compartment compared to the whole kidney. However, 25 genes were differentially expressed in the glomerular compartment compared to the whole kidney. Our data showed that sufficient RNA can be extracted from small compartments like mouse renal glomeruli from archival FFPE tissue, and that whole kidney analysis does not accurately represent the transcriptome state of the glomeruli, which comprise only a small proportion of the overall kidney volume
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    Relationship of human papillomavirus with seborrheic keratosis of the female genital tract - a case-series and literature review
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Dasgupta, Shatavisha; Eersel, Rachel van; Morrel, Beth; Munckhof, Henk A.M. van den; Geus, Vera A. de; Hoeven, Nick M.A. van der; Sandt, Miekel M. van de; Piso-Jozwiak, Marta; Quint, Wim G.V.; Avoort, Irene A.M. van der; Koljenović, Senada; Ewing-Graham, Patricia C.; Kemenade, Folkert J. van
    Seborrheic keratoses (SKs) are benign lesions of uncertain etiology, which can develop in both genital and extra-genital locations. For genital SKs there has been conjecture about the pathogenic role of human papillomavirus (HPV), in view of the frequent association of this virus with genital lesions. In light of the potential consequences on patient management, we investigated the relationship between HPV and SKs of the female genital tract (FGT). For this, we evaluated the current evidence on this relationship by performing an in-depth review of the literature. Furthermore, to add to the evidence on this association, we investigated the presence of HPV in a series of vulvar SKs (n=15), using a novel multimodal approach. This involved whole tissue section-polymerase chain reaction (WTS-PCR) using SPF10-DEIA-LiPA25 for HPV detection and genotyping. In addition, immunohistochemistry (IHC) was performed with cellular biomarkers p16 and MIB-1, and viral biomarker E4, to augment HPV-testing. Finally, laser-capture microdissection-PCR (LCM-PCR) was performed to locate HPV to specific lesional cells, and to rule out incidental detection of resident HPV with WTS-PCR. Our findings from the literature review as well as the case-series are presented.