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ABSTRACT Studies on public health and wild mammal biodiversity include a genetic component. For blood samples, there must be optimal sample collection conditions since these can affect DNA preservation and extraction. This study evaluated the use of liquid and dry DNA preservation methods and commercial and non-commercial DNA extraction methods on field-collected blood samples. For this, 264 total blood samples were collected from wild mammals. A first group of samples was preserved in guanidine hydrochloride (GuHCl) and DNA was extracted using six commercial kits: Bioline, Norgen, Invitrogen, Promega, and Qiagen, in addition to phenol-chloroform isoamyl alcohol (PC) and guanidine thiocyanate (GIT). Another group of samples was preserved in Whatman® FTA® cards and DNA was extracted with PC and GIT. The extractions with GIT and PC showed the highest values (ng/µL) and variation in DNA concentration, while the commercial kit showed low variation. Sample preservation in Whatman® FTA® cards provided low variation and quantity of the extracted DNA compared with the use of GuHCl. Concerning DNA quality, the commercial kits yielded higher purity, while GIT and PC-based protocols provided highly variable results. Furthermore, the use of GIT and PC yielded a higher amount of DNA, yet, of variable quality. Overall, extraction based on commercial kits and Whatman® FTA® preservation allowed obtaining more standardized DNA qualities and quantities.
RESUMEN Los estudios sobre salud pública y biodiversidad de mamíferos silvestres incluyen un componente genético. Para las muestras de sangre, se debe tener condiciones óptimas de colección, ya que pueden afectar la preservación y la extracción del ADN. Este estudio evaluó el uso de métodos de preservación de ADN líquido y seco y métodos de extracción de ADN comerciales y no comerciales, en muestras de sangre, recolectadas en campo. Para ello, se recogieron 264 muestras de sangre totales de mamíferos salvajes. Se preservó un primer grupo de muestras en clorhidrato de guanidina (GuHCl) y se extrajo el ADN, utilizando seis kits comerciales: Bioline, Norgen, Invitrogen, Promega y Qiagen, además de dos protocolos no comerciales: fenol-cloroformo isoamil alcohol (PC) y guanidina tiocianato (GIT). Otro grupo de muestras, se preservó en tarjetas Whatman® FTA® y se extrajo el ADN, con PC y GIT. Las extracciones con GIT y PC mostraron los valores y variaciones más altas en la concentración de ADN (ng/µL), mientras que el kit comercial mostró una baja variación. La preservación de la muestra en tarjetas Whatman® FTA® proporcionó una baja variación y cantidad de ADN extraído, en comparación con el uso de GuHCl. En cuanto a la calidad del ADN, los kits comerciales produjeron una mayor pureza (A260/280), mientras que los protocolos basados en GIT y PC proporcionaron resultados muy variables. Además, el uso de GIT y PC originó una mayor cantidad de ADN, pero de calidad variable. En general, la extracción basada en kits comerciales y la conservación Whatman® FTA® permitió obtener calidades y cantidades de ADN más estandarizadas.
ABSTRACT
Objective The purpose of this study was to evaluate the quality of DNA from the formalin-fixed paraffin-embedded (FFPE) specimens of non-small cell lung cancer (NSCLC) after immunohistochemical staining and investigate DNA extraction by immunohistochemical staining of the specimens in small in number or difficult to obtain and the feasibility of related molecular tests. Methods We randomly collected 50 FFPE biopsy specimens of NSCLC in our Department of Pathology from June 2017 to December 2017 and sliced each into 12 sections, of which, 6 were directly subjected to DNA extraction (the control group) and the other 6 to immunohistochemical Envision two-step staining for DNA extraction (the experimental group). Then, we detected the mutations of the epidermal growth factor receptor (EGFR), kirsten rat sarcoma viral oncogene (KRAS) and V-raf murine sarcoma viral oncogene homolog B (BRAF) in all the DNAs extracted. Results No statistically significant differences were observed between the experimental and control groups in the DNA concentration and purity in the 50 FFPE biopsy specimens of NSCLC (P>0.05). Of the 50 NSCLC FFPE specimens of the experimental group, 20 (40%) showed the mutation of EGFR, 8 (16%) exhibited that of KRAS, and 5 (10%) manifested that of BRAF. In the other 50 specimens of the control group, 33 showed the mutations of EGFR, KRAS and BRAF. A 100% consistency was found in the results of detection between the experimental and control groups (P=0.000, Kappa=1.000). Conclusion High-quality DNA can be extracted after immunohistochemical staining from NSCLC FFPE specimens, especially those small in number or difficult to obtain, and can be used for downstream molecular analysis of target genes, which is a good method for specimen recycling and provides a solution for subsequent molecular test of scarce or difficult-to-obtain clinical samples.
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Aims@#The application of mouthwash is one of the oral hygiene treatments that commonly use after tooth brushing to control the bacterial colonization from overgrowth. This research is focused on investigating the effect of mouthwash on oral microbiome by analyzing the quality and yield of DNA obtained before and after using mouthwash and also to compare the bacterial abundance via 16S rRNA PCR detection.@*Methodology and results@#The DNA was extracted from the saliva samples before and after using mouthwash using Phenol-Chloroform extraction method. The DNA extract was then evaluated using Nano Drop ND-1000 UV/VIS Spectrophotometer to determine the DNA quality and DNA yield. After that, the 16S rRNA gene was amplified via PCR for bacterial detection in the saliva using 27 F and 1492 R primers set, and the PCR products were observed on 1.5% gel electrophoresis. Statistical analysis was performed by using Graphpad Prism 7.03 software. For DNA yield, there was significantly higher yield observed after mouthwash usage with 80% of the samples was found to yield more DNA. To assess DNA quality, absorbance ratio of A260/A280 and A260/A230 were used. The DNA quality was seen to be similar for both A260/A280 and A260/A230 absorbance ratio even after the usage of mouthwash. The amplification of 16S rRNA gene was successful and 1500 bp expected band size was observed.@*Conclusion, significance and impact of study@#This study demonstrated the usage of mouthwash is useful to increase the DNA yield as compared to without using mouthwash. However in terms of quality, no difference is seen. This result can be used to provide insight on mouthwash usage for saliva sampling in a non-invasive manner.
ABSTRACT
DNA extraction methods were evaluated for the yield and purity of DNA recovered from mycorrhized roots and whether the recovered DNA is suitable for amplification of arbuscular mycorrhizal (AM) fungal SSU rDNA. The DNeasy Plant Mini Kit and three extraction buffers were used alone or in combination with either polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP) and/or activated charcoal (AC). Among the extraction methods tested, those based on the CTAB buffers yielded more DNA than those based on the TE buffer and the DNeasy Plant Mini Kit. Moreover, the use of AC alone or in combination with PVPP reduced DNA yield, while it significantly improved the purity of recovered DNA, whatever the extraction buffer. On the other hand, the success of nested-PCR amplification was negatively correlated with the amount of template DNA and positively correlated with the purity of recovered DNA. Three methods based on the TE buffer, two on the CTAB-ßM buffer and one on the DNeasy Plant Mini Kit produced high-quality DNA in terms of purity and PCR performance. However, the TE buffer-based methods are less time consuming than the CTAB-ßM buffer-based methods, and cheaper than the method based on the DNeasy Plant Mini Kit.
Diferentes métodos de extração de ADN, a partir de amostras de raízes, foram testados e o ADN obtido foi avaliado para a amplificação de genes ribossomais de fungos micorrízicos arbusculares (AM). Três tampões de extração foram utilizados isoladamente ou em combinação com polivinilpirrolidona (PVP), polivinipolipirrolidona (PVPP) e/ou carvão ativado (CA), além do DNeasy Plant Mini Kit. Entre os métodos de extração testados, aqueles com base no tampão CTAB renderam mais ADN do que os baseados no tampão TE e o DNeasy Plant Mini Kit. A utilização de CA ou PVPP nos diferentes tampões reduziram o rendimento de ADN, contudo, melhoraram significativamente a pureza do ADN recuperado, independentemente do tampão de extração. Por outro lado, o sucesso da amplificação por Nested-PCR foi negativamente correlacionado com a quantidade de DNA molde, e positivamente correlacionada com a pureza do ADN. Três métodos baseados no tampão TE, dois no tampão CTAB-ßM e o DNeasy Plant Mini Kit produziram ADN de alta qualidade, em termos de pureza e rendimento da PCR. No entanto, os métodos baseados em tampão TE demandam menos tempo do que os métodos baseados em tampão CTAB-ßM e são mais baratos do que o uso do DNeasy Plant Mini Kit.