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.

scholarly journals The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use

2021 ◽  
Author(s):  
Robin Verjans ◽  
Annette H. Bruggink ◽  
Robby Kibbelaar ◽  
Jos Bart ◽  
Aletta Debernardi ◽  
...  
Keyword(s):  
The Netherlands ◽  
Tissue Sample ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Internet Portal ◽  
Formalin Fixed Paraffin ◽  
Practical Support ◽  
Formalin Fixed Paraffin Embedded ◽  
The Rich ◽  
Formalin Fixed

AbstractBiobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation—a nationwide network and registry of histo- and cytopathology in the Netherlands—was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA’s nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use.  We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA’s nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (− 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA’s national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands.

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.

scholarly journals Deciphering the Immune Microenvironment on A Single Archival Formalin-Fixed Paraffin-Embedded Tissue Section by An Immediately Implementable Multiplex Fluorescence Immunostaining Protocol

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2449 ◽  
Author(s):  
Adrien Guillot ◽  
Marlene S. Kohlhepp ◽  
Alix Bruneau ◽  
Felix Heymann ◽  
Frank Tacke
Keyword(s):  
Immune Cell ◽  
Simple Procedure ◽  
Parenchymal Cell ◽  
Ffpe Tissue ◽  
Immune Microenvironment ◽  
Tissue Samples ◽  
Tissue Organization ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Technological breakthroughs have fundamentally changed our understanding on the complexity of the tumor microenvironment at the single-cell level. Characterizing the immune cell composition in relation to spatial distribution and histological changes may provide important diagnostic and therapeutic information. Immunostaining on formalin-fixed paraffin-embedded (FFPE) tissue samples represents a widespread and simple procedure, allowing the visualization of cellular distribution and processes, on preserved tissue structure. Recent advances in microscopy and molecular biology have made multiplexing accessible, yet technically challenging. We herein describe a novel, simple and cost-effective method for a reproducible and highly flexible multiplex immunostaining on archived FFPE tissue samples, which we optimized for solid organs (e.g., liver, intestine, lung, kidney) from mice and humans. Our protocol requires limited specific equipment and reagents, making multiplexing (>12 antibodies) immediately implementable to any histology laboratory routinely performing immunostaining. Using this method on single sections and combining it with automated whole-slide image analysis, we characterize the hepatic immune microenvironment in preclinical mouse models of liver fibrosis, steatohepatitis and hepatocellular carcinoma (HCC) and on human-patient samples with chronic liver diseases. The data provide useful insights into tissue organization and immune–parenchymal cell-to-cell interactions. It also highlights the profound macrophage heterogeneity in liver across premalignant conditions and HCC.

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
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