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Quorum Sensing and Essential Oils
Published in K. Hüsnü Can Başer, Gerhard Buchbauer, Handbook of Essential Oils, 2020
Isabel Charlotte Soede, Gerhard Buchbauer
Sharifi et al. (2018) used RT-PCR to detect QS inhibition in Staphylococcus aureus strains by detecting expressional changes of QS genes (RNAIII). To do so, biofilms were grown with and without EOs. RNA was then isolated by using commercial RNA extraction and purification kits. RNA was then reverse transcribed to cDNA. This cDNA was used for quantitative real-time PCR to detect expression levels of RNAIII. Primer pairs used for RT-PCR assay are, for example, hld gene (delta-hemolysin gene), which is part of the RNAIII gene and 16S (rRNA) (Sharifi et al., 2018).
Tissue is the Issue
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
Fresh tissue. Nonfrozen and nonfixed tissue should be collected directly into a labeled sterile screw-cap plastic container, with one of the following buffers: Phosphate buffered saline (PBS) for tissue culture and cell viability studies.Neutral buffered formalin (10%) for gradual fixation.RPMI (Roswell Park Memorial Institute) media for tissue culture, cell viability studies, protein extraction, and multiphoton studies.RNAlater for RNA extraction.OCT bottom and top prior to freezing.
Companion Diagnostic (CDx) Tests in Clinical Laboratory Improvement Amendments (CLIA)-Certified Laboratories
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
DNA and/or RNA extraction: Once the tissue status is checked, DNA and/or RNA are extracted according to the SOPs. The second most common reason for CLIA NGS testing failure is insufficient DNA (28.9% of total failed cases).17 Thus, it is critical to efficiently extract DNA first and then correctly quantify the amount.
Development of an effective and rapid qPCR for identifying human ChREBPα/β isoforms in hepatic and adipose tissues
Published in Scandinavian Journal of Clinical and Laboratory Investigation, 2019
Sandra M. Ramírez-Meza, Montserrat Maldonado-González, Zamira H. Hernández-Nazara, Erika Martínez-López, Saúl Ocampo-González, Lucina Bobadilla-Morales, José R. Torres-Baranda, Bertha Ruíz-Madrigal
In the present study, a qPCR assay based on the hydrolysis probes was developed to detect both ChREBP isoforms. This qPCR assay was designed to meet the key parameters of the MIQE guidelines [15]. Among the requirements is the quality of the input nucleic acid. Accordingly, RNA extraction is a crucial step for monitoring gene expression. Poor RNA quality (including degradation and remaining impurities) can result in misleading results. Particularly, isolation of RNA from high lipid content or collagen-rich tissues can be challenging [20,21]. In addition, it is recommended that when the extraction of total RNA from adipose tissue is carried out, the modifications proposed above should be performed, since the quality (260 nm/280 nm ration >1.8) and yield (approximately 26% more) of the total RNA obtained are greatly improved.
Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next-generation sequencing
Published in Journal of Extracellular Vesicles, 2018
Dominik Buschmann, Benedikt Kirchner, Stefanie Hermann, Melanie Märte, Christine Wurmser, Florian Brandes, Stefan Kotschote, Michael Bonin, Ortrud K. Steinlein, Michael W. Pfaffl, Gustav Schelling, Marlene Reithmair
One of the most important aspects of EV research is analysing their nucleic acid cargo, particularly small RNAs. These are commonly quantified by RT-qPCR or, increasingly, comprehensive transcriptomic profiling by next-generation sequencing (NGS, small RNA-Seq). Applications of EV transcriptomics range from basic research to biomarker discovery and drug development, making use of EVs as an easily accessible, enriched sampling fraction [2,3]. Inferring credible information from the transcriptome relies on precise quantification of target RNA, which in turn requires samples of high quality and integrity [4]. Additionally, methods for RNA extraction itself influence downstream analyses by yielding non-identical, kit-specific isolates [5]. This holds true particularly for extracellular RNA, which bears additional challenges such as low concentrations, diminished RNA integrity and high variability between individuals. Indeed, recent publications have highlighted the impact of cell-free RNA extraction strategies on small RNA-Seq, reporting quantitative and qualitative differences in resulting sequencing libraries [6,7].
A technical assessment of the porcine ejaculated spermatozoa for a sperm-specific RNA-seq analysis
Published in Systems Biology in Reproductive Medicine, 2018
Marta Gòdia, Fabiana Quoos Mayer, Julieta Nafissi, Anna Castelló, Joan Enric Rodríguez-Gil, Armand Sánchez, Alex Clop
Two determinant parameters for a successful transcriptome analysis are both the RNA quality and yield. The RNA extraction method becomes a critical step when working with spermatozoa, since these cells have low amount of highly fragmented RNA. In the present work, we chose the Trizol method for RNA extraction after having tested other protocols involving commercial kits, which yielded even lower RNA yields (data not shown). The average amount of RNA extracted per sperm cell was 1.6 fg, a similar value to previously reported data in domestic swine (Yang et al. 2009), but lower than human (Pessot et al. 1989; Goodrich et al. 2013) and mice (Pessot et al. 1989). The low amount of RNA recovered and low RIN value is in fact an indication that the removal of somatic cells, with their large amount of non-fragmented RNA, during the cell purification steps, was highly efficient. The observed variability in RNA yields between samples could be due to inter-sample differences in the epididymosomes secreted by epididymal epithelial cells, which have been involved in post-testicular spermatogenesis and are known to contain a repertoire of RNAs (Belleannée et al. 2013), yet this mechanism remains to be elucidated.