scholarly journals Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

2016 ◽  
Vol 54 (11) ◽  
pp. 2798-2803 ◽  
Author(s):  
Elham Salehi ◽  
Mohammad T. Hedayati ◽  
Jan Zoll ◽  
Haleh Rafati ◽  
Maryam Ghasemi ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Aspergillus Flavus ◽  
Ffpe Tissue ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4- to 5-μm FFPE tissue sections from each specimen were digested with proteinase K, followed by automated nucleic acid extraction. Multiple real-time quantitative PCR (qPCR) assays targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, using fluorescently labeled primers, was performed to identify clinically important genera and species of Aspergillus , Fusarium , Scedosporium , and the Mucormycetes . The molecular identification was correlated with results from histological examination. One of the main findings of our study was the high sensitivity of the automated DNA extraction method, which was estimated to be 94%. The qPCR procedure that was evaluated identified a range of fungal genera/species, including Aspergillus fumigatus , Aspergillus flavus , Aspergillus terreus , Aspergillus niger , Fusarium oxysporum , Fusarium solani , Scedosporium apiospermum , Rhizopus oryzae , Rhizopus microsporus , Mucor spp., and Syncephalastrum . Fusarium oxysporum and F. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis. Aspergillus flavus , S. apiospermum , and Syncephalastrum were detected from histopathological mucormycosis samples. In addition, examination of four samples from patients suspected of having concomitant aspergillosis and mucormycosis infections resulted in the identification of two A. flavus isolates, one Mucor isolate, and only one sample having both R. oryzae and A. flavus . Our results indicate that histopathological features of molds may be easily confused in tissue sections. The qPCR assay used in this study is a reliable tool for the rapid and accurate identification of fungal pathogens to the genus and species levels directly from FFPE tissues.

Copper adhesive tape attached to reverse side of non-conductive glass slide to achieve protein MALDI-imaging in FFPE-tissue sections

2021 ◽  
Author(s):  
Ran Wu ◽  
Liang Qin ◽  
Lulu Chen ◽  
Rui Ma ◽  
Difan Chen ◽  
...  
Keyword(s):  
Ffpe Tissue ◽  
Glass Slide ◽  
Maldi Imaging ◽  
Adhesive Tape ◽  
Tissue Sections ◽  
Conductive Glass ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Herein, copper adhesive tape attached to reverse side of glass slide was developed as a new method to achieve protein in-situ detection and imaging in the formalin fixed paraffin-embedded (FFPE)...

scholarly journals Tissue lithography: Microscale dewaxing to enable retrospective studies on formalin-fixed paraffin-embedded (FFPE) tissue sections

PLoS ONE ◽  
2017 ◽  
Vol 12 (5) ◽  
pp. e0176691 ◽  
Author(s):  
Julien F. Cors ◽  
Aditya Kashyap ◽  
Anna Fomitcheva Khartchenko ◽  
Peter Schraml ◽  
Govind V. Kaigala
Keyword(s):  
Retrospective Studies ◽  
Ffpe Tissue ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Quantitative determination of predictive cancer biomarkers in formalin-fixed, paraffin-embedded tissue using a new, fully automated method for RNA isolation

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 11032-11032
Author(s):  
B. M. Müller ◽  
R. Kronenwett ◽  
G. Hennig ◽  
K. Weber ◽  
K. Bohmann ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Cancer Biomarkers ◽  
Ffpe Tissue ◽  
Rt Pcr ◽  
Total Rna ◽  
Tissue Sections ◽  
Automated Method ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

11032 Background: Personalized cancer therapy depends on the evaluation of tissue-based biomarkers in routine tumor samples. As many new biomarkers are measured on the mRNA level, standardized procedures for mRNA analysis in formalin-fixed, paraffin-embedded (FFPE) tissue are needed. In this study, we present a novel, standardized, and fully automated method for fast isolation of total RNA from FFPE tissue sections. Methods: Tissue samples (n = 501) from 167 breast carcinomas which had been stored between 2 months and 21 years were investigated. Total RNA was extracted from tissue sections using a new method based on silica-coated iron oxide beads in combination with a specific liquid-handling robot. Novel and unique features of the method are an automated extraction-integrated deparaffinization step and a bead-based negative selection step to eliminate any undigested tissue. Yield of RNA was assessed using the Ribogreen assay. mRNA fragment lengths were estimated by reverse transcription PCR (RT-PCR) for G6PDH. Expression of the breast cancer biomarkers ESR1, PGR and HER2 was measured by kinetic RT-PCR (kPCR) and compared with immunohistochemistry (IHC). Results: RNA was successfully isolated from all samples, with a mean yield of 1.4 μg/sample (range: 0.1–7.8 μg) and fragment lengths of at least 150 bp in 99% of samples. PCR analyses could be performed in all samples. Investigating three sections of each tumor, we observed a low section-to-section variability of kPCR results (root of mean squared errors of relative ESR1, PGR or HER2 expression in three sections: 0.2–0.5 Ct values). Comparing kPCR results with IHC, we detected a good concordance between both methods, with agreements of 98.4% (standard error [SE]: 2.7%) for ESR1, 84.4% (SE: 5.3%) for PGR and 89.9% (SE: 8.9%) for HER2. Conclusions: This novel RNA extraction method is a major technical improvement for implementation of reproducible, high-throughput and cost-efficient testing of cancer biomarkers in the clinical routine and in gene-expression research studies using archived FFPE material in molecular labs. [Table: see text]

Evaluation of the efficacy of various reagents in improving microRNA extraction

2019 ◽  
Vol 56 (3) ◽  
pp. 375-380
Author(s):  
Arizumi Kikuchi ◽  
Azumi Naruse ◽  
Takahiro Sawamura ◽  
Kenichi Nonaka
Keyword(s):  
Ffpe Tissue ◽  
Tetraethylene Glycol ◽  
Optimum Amount ◽  
Stem Loop ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Crossing Point ◽  
Formalin Fixed

Background MicroRNA has received considerable attention in the clinical context, and attempts are being made to use microRNA in clinical diagnosis. However, adequate quantities of microRNA required for analysis are challenging to isolate. We tested the effect of various reagents in improving microRNA extraction and compared their efficacy to that of a commercially available extraction kit (HighPure miRNA isolation kit, Roche). Methods We used the synthetic oligonucleotide miR-21 and formalin-fixed, paraffin-embedded (FFPE) tissue sections from colon cancer samples ( n = 10). We tested increasing volumes (100–600  μL) of 1,4-dioxane, 2-butanol, 2-propanol, acetonitrile, polyethylene glycol (PEG) 600, PEG 1000, PEG 1540, PEG 2000, tetraethylene glycol dimethyl ether (TDE), and tetrahydrofuran, instead of the binding enhancer solution provided in the kit. MiR-21 analysis was performed via stem-loop RT-qPCR using Universal ProbeLibrary probe (Roche). Results The optimum amount of each enhancement solution was 200–500  μL. We obtained ΔCp values of optimum additional volume for each solution from 1.04 to 2.50 and compared these with those obtained using the commercially available kit. PEG 1540 and 2000 produced superior reactivity with minimal addition. For FFPE tissue samples, addition of the enhancement solutions PEG 1540 and 2000 resulted in mean crossing point values of 18.15 ± 2.26 and 17.73 ± 3.26, respectively. We obtained a crossing point value of 20.56 ± 4.26 (mean ± SD) using the commercially available kit. Conclusions The tested enhancer reagents, which are relatively readily available and easy to use, can improve microRNA extraction efficacy of a commercially available kit.

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