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In samples of harmful algal blooms (HABs), seawater can contain a high abundance of microorganisms and elemental ions. Along with the hardness of the walls of key HAB dinoflagellates such as Prorocentrum triestinum, this makes RNA extraction very difficult. These components interfere with RNA isolation, causing its degradation, in addition to the complex seawater properties of HABs that could hinder RNA isolation for effective RNA sequencing and transcriptome profiling. In this study, an RNA isolation technique was established through the modification of the Trizol method by applying the Micropestle System on cell pellets of P. triestinum frozen at -20 °C, obtained from 400 mL of culture with a total of 107 cells/mL. The results of the modified Trizol protocol generated quality RNA samples for transcriptomics sequencing, as determined by their measurement in Analyzer Agilent 4150.

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INTRODUCTION: High-quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim. OBJECTIVE: To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol. METHODOLOGY: Based on the conventional Trizol method, the modified Trizol method 1 and modified Trizol method 2 were used as the control for extraction of RNA from E. senticosus Maxim leaves. The modified Trizol method 1 added ß-mercaptoethanol on the conventional Trizol method. After RNA was dissolved, a mixed solution of phenol, chloroform, and isoamyl alcohol was added to denature protein and inhibit the degradation of RNA. The modified Trizol method 2 adds PVPP to grind on the basis of modified Trizol method 1, so as to better remove phenols from leaves, and eliminates the step of incubation at -20°C to reduce extraction time and RNA degradation. Chloroform, CTAB, and CH3COONa were used instead of a phenol, chloroform, and isoamyl alcohol mixed solution to ensure complete separation of nucleic acid from plant tissues and to obtain high-purity RNA. RESULTS: The research results showed that the quality of RNA extracted by conventional Trizol method, modified Trizol method 1, was incomplete, accompanied with different degrees of contamination of polysaccharides, polyphenols, and DNA. The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. The ratio of A260/A280 was in the range of 1.8-2.0, and the yield of RNA was the highest, which was 1.68 and 1.15 times compared with that by conventional Trizol method and modified Trizol method 1 extraction, respectively. The reverse transcription cDNA was further tested through PCR with the specific primers. The amplified fragments are displayed in clear and bright bands in accordance with the expected size. CONCLUSION: The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. High-quality RNA has more advantages in molecular biology study of E. senticosus Maxim.

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Since the development of next-generation sequencing techniques and with the growing interest in transcriptomic studies, there is a demand for high-throughput RNA extraction techniques. General RNA extraction protocols are unreliable when it comes to the quality and quantity of isolated RNA obtained from different tissue types of different plant species. Despite Norway spruce (Picea abies) being one of the most significant and commercially valuable tree species in European forests, only limited genetic research is available. In this study, we developed a cetyltrimethylammonium bromide (CTAB) protocol by modifying the original method. We compared this CTAB protocol with other widely used methods for extracting RNA from different tissues (needle, phloem, and root) of Norway spruce, known for its richness in polyphenols, polysaccharides, and secondary metabolites. The modified CTAB method proves to be superior to the kit-based and TRIzol-based methods for extracting RNA from the metabolite-rich tissues of Norway spruce, resulting in high RNA quality and integrity values (RIN~7-9). The modified CTAB RNA extraction method is rapid, cost-effective, and relatively simple in yielding the desired RNA quality from Norway spruce tissues. It is optimal for RNA sequencing and other downstream molecular applications.

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Increasing evidence of sperm RNA's role in fertilization and embryonic development has provided impetus for its isolation and thorough characterization. Sperm are considered tough-to-lyse cells due to the compact condensed DNA in sperm heads. Lack of consensus among bovine sperm RNA isolation protocols introduces experimental variability in transcriptome studies. Here, we describe an optimized method for total RNA isolation from bovine sperm using the TRIzol reagent. This study critically investigated the effects of various lysis conditions on sperm RNA isolation. Sperm suspended in TRIzol were subjected to a combination of mechanical treatments (sonication and passage through a 30G needle and syringe) and chemical treatments (supplementation with reducing agents 1,4-dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride (TCEP)). Microscopic evaluation of sperm lysis confirmed preferential sperm tail versus sperm head lysis. Interestingly, only TCEP-supplemented TRIzol (both mechanical treatments) had progressive sperm head lysis and consistently yielded total sperm RNA. Furthermore, RNA integrity was confirmed based on the electrophoresis profile and an absence of genomic DNA and somatic cells (e.g., epithelial cells, spermatids, etc.) with RT-qPCR. Our findings highlighted the importance of sperm lysis, specifically of the sperm head using TCEP with mechanical treatment, in total RNA isolation and presented a bovine-specific sperm RNA isolation method to reduce experimental variabilities.

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Procuring high-grade RNA from mature coconut tissues is a tricky and labor-intensive process due to the intricate scaffold of polysaccharides, polyphenols, lipids, and proteins that form firm complexes with nucleic acids. However, we have effectively developed a novel method for the first time, letting the retrieval of high-grade RNA from the roots, endosperm, and mesocarp of mature coconut trees take place. In this method, we exploited dichloromethane as a replacement to phenol/chloroform for RNA recovery from mature coconut tissues. The amount of high-grade RNA acquired from the roots of mature coconut trees was 120.7 µg/g, with an A260/280 ratio of 1.95. Similarly, the mature coconut mesocarp yielded 134.6 µg/g FW of quality RNA with A260/280 ratio of 1.98, whereas the mature coconut endosperm produced 120.4 µg/g FW of quality RNA with A260/280 ratio of 2.01. Furthermore, the RNA isolation using the dichloromethane method exhibited excellent performance in downstream experiments, particularly in RT-PCR for cDNA production and amplification. On the contrary, the RNA plant kit, TRIZOL, and Cetyl Trimethyl Ammonium Bromide (CTAB) methods were unsuccessful in isolating substantial quantities of RNA with exceptional purities from the mentioned coconut tissues. In view of these findings, we conclude that the newly developed method will be pivotal in effectively extracting RNA with high purity from mature coconut tissues.

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BACKGROUND: High-purity RNA serves as the basic requirement for downstream molecular analysis of plant species, especially the differential expression of genes to various biotic and abiotic stimuli. But, the extraction of high-quality RNA is usually difficult from plants rich in polysaccharides and polyphenols, and their presence usually interferes with the downstream applications. The aim of the study is to optimize the extraction of high-quality RNA from diverse plant species/tissues useful for downstream molecular applications. RESULTS: Extraction of RNA using commercially available RNA extraction kits and routine hexadecyltrimethylammonium bromide (CTAB) methods did not yield good quality DNA-free RNA from Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera. A reliable protocol for the extraction of high-quality RNA from mature leaves of these difficult-to-extract trees was optimized after screening nine different methods. The DNase I-, and proteinase K treatment-free modified method, consisting of extraction with CTAB method followed by TRIzol, yielded high-quality DNA-free RNA with an A260/A280 and A260/A230 ratios > 2.0. Extraction of RNA from Conocarpus, the most difficult one, was successful by avoiding the heat incubation of ground tissue in a buffer at 65 oC. Pre-warming of the buffer for 5-10 min was sufficient to extract good-quality RNA. RNA integrity number of the extracted RNA samples ranged between 7 and 9.1, and the gel electrophoresis displayed intact bands of 28S and 18S RNA. A cDNA library constructed from the RNA of P. cineraria was used for the downstream applications. Real-time qPCR analysis using the cDNA from P. cineraria RNA confirmed the quality. The extraction of good quality RNA from samples of the desert-growing P. cineraria (> 20-years-old) collected in alternate months of the year 2021 (January to December covering winter, spring, autumn, and the very dry and hot summer) proved the efficacy of the protocol. The protocol's broad applicability was further validated by extracting good-quality RNA from 36 difficult-to-extract plant species, including tissues such as roots, flowers, floral organs, fruits, and seeds. CONCLUSIONS: The modified DNase I and Proteinase K treatment-free protocol enables to extract DNA-free, high-quality, intact RNA from a total of 39 difficult-to-extract plant species belonging to 32 angiosperm families is useful to extract good-quality RNA from dicots and monocots irrespective of tissue types and growing seasons.

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High yield and integrity of placental RNA are crucial for placental transcriptomics studies. We assessed the effects of time to placental collection post-delivery; tissue storage, amount and method used for extraction; mode of delivery; and tissue type on total RNA yield. The optimal protocol for RNA extraction from placental tissue includes cryofreezing of the sample upon collection and RNA extraction from 50 mg of tissue using TRIzol reagent. Decidua yielded highest RNA quantity/mg of tissue, followed by villous tissue and the chorion. Comparisons with murine kidney and HEK293T show lower placental RNA yield, likely due to highly dense and heterogeneous tissue make-up and potential high placental nuclease activity.

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The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories.

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High-quality RNA isolation from recalcitrant adipose tissue with high lipid content and low cell numbers is difficult. Many studies have made efforts to optimize methods for isolating RNA from adipose tissue through combinations of column-based kits and phenol-chloroform methods, or through in-house protocols. However, the considerable complexity of these protocols and the various kits/materials required hamper their wide use. Herein, we describe an optimized protocol based on TRIzol reagent, which is the most accessible ready-to-use reagent for nucleic acid and/or protein isolation in laboratories. This article provides a step-by-step protocol yielding sufficient and qualified RNA from lipid-rich specimens for downstream applications.

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Articular cartilage is characterized by a low density of chondrocytes surrounded by an abundant extracellular matrix (ECM) consisting of a dense mixture of collagens, proteoglycans, and glycosaminoglycans. Due to its low cellularity and high proteoglycan content, it is particularly challenging to extract high-quality total RNA suitable for sensitive high-throughput downstream applications such as RNA sequencing (RNA-Seq). Available protocols for high-quality RNA isolation from articular chondrocytes are inconsistent, resulting in suboptimal yield and compromised quality. This poses a significant difficulty in the application of RNA-Seq to study the cartilage transcriptome. Current protocols rely either on dissociation of cartilage ECM by collagenase digestion or pulverizing cartilage using various methods prior to RNA extraction. However, protocols for cartilage processing vary significantly depending on the species and source of cartilage within the body. Protocols for isolating RNA from human or large mammal (e.g., horse or cattle) cartilage samples are available, but this is not the case for chicken cartilage, despite the species being extensively used in cartilage research. Here, we present two improved RNA isolation protocols based on pulverization of fresh articular cartilage using a cryogenic mill or on enzymatic digestion using 1.2% (w/v) collagenase II. Our protocols optimize the collection and tissue processing steps to minimize RNA degradation and enhance RNA purity. Our results show that RNA purified from chicken articular cartilage using these methods has appropriate quality for RNA-Seq experiments. The procedure is applicable for RNA extraction from cartilage from other species such as dog, cat, sheep, and goat. The workflow for RNA-Seq analysis is also described here. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of total RNA from pulverized chicken articular cartilage Alternate Protocol: Extraction of total RNA from collagen-digested articular cartilage Support Protocol: Dissection of chicken articular cartilage from the knee joint Basic Protocol 2: RNA sequencing of total RNA from chicken articular cartilage.

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Simultaneous extraction of various types of biomolecule from a single sample can be beneficial for multiomics studies of unique specimens. An efficient and convenient sample preparation approach must be developed that can comprehensively isolate and extract biomolecules from one sample. TRIzol reagent is widely used in biological studies for DNA, RNA, and protein isolation. This study evaluated the feasibility of using TRIzol reagent for the simultaneous isolation of not only DNA, RNA, and proteins but also metabolites and lipids from a single sample. Through the comparison of known metabolites and lipids obtained using the conventional methanol (MeOH) and methyl-tert-butyl ether (MTBE) extraction methods, we determined the presence of metabolites and lipids in the supernatant during TRIzol sequential isolation. Finally, we performed untargeted metabolomics and lipidomics to examine metabolite and lipid alterations associated with the jhp0417 mutation in Helicobacter pylori by using the TRIzol sequential isolation protocol and MeOH and MTBE extraction methods. Metabolites and lipids with significant differences isolated using the TRIzol sequential isolation protocol were consistent with those obtained using the conventional MeOH and MTBE extraction methods. These results indicated that TRIzol reagent can be used to simultaneously isolate metabolites and lipids from a single sample. Thus, TRIzol reagent can be used in biological and clinical research, especially in multiomics studies.

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BACKGROUND: A mixture of phenol and guanidine isothiocyanate ("P/GI", the principal components of TRIzol™ and similar products) is routinely used to isolate RNA, DNA, and proteins from a single specimen. In time-course experiments of cells grown in tissue culture, replicate wells are often harvested sequentially and compared, with the assumption that in-well lysis and complete aspiration of P/GI has no effect on continuing cultures in nearby wells. METHODS: To test this assumption, we investigated morphology and function of RAW 264.7 cells (an immortalized mouse macrophage cell line) cultured in covered 96-well plates for 4, 8, or 24 h at varying distances from a single control well or a well into which P/GI had been deposited and immediately aspirated completely. RESULTS: Time- and distance-dependent disruptions resulting from proximity to a single well containing trace residual P/GI were seen in cell morphology (blebbing, cytoplasmic disruption, and accumulation of intracellular vesicles), cell function (pH of culture medium), and expression of genes related to inflammation (Tnfα) and autophagy (Lc3b). There was no transcriptional change in the anti-apoptotic gene Mcl1, nor the pro-apoptotic gene Hrk, nor in P/GI-unexposed control cultures. LPS-stimulated cells incubated near P/GI had lower expression of the cytokine Il6. These effects were seen as early as 4 h of exposure and at a distance of up to 3 well units from the P/GI-exposed well. CONCLUSIONS: Exposure to trace residual quantities of P/GI in covered tissue culture plates leads to substantial disruption of cell morphology and function in as little as 4 h, possibly through induction of autophagy but not apoptosis. This phenomenon should be considered when planning time-course experiments in multi-well covered tissue culture plates.

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Safe sample transport is of great importance for infectious diseases diagnostics. Various treatments and buffers are used to inactivate pathogens in diagnostic samples. At the same time, adequate sample preservation, particularly of nucleic acids, is essential to allow an accurate laboratory diagnosis. For viruses with single-stranded RNA genomes of positive polarity, such as foot-and-mouth disease virus (FMDV), however, naked full-length viral RNA can itself be infectious. In order to assess the risk of infection from inactivated FMDV samples, two animal experiments were performed. In the first trial, six cattle were injected with FMDV RNA (isolate A22/IRQ/24/64) into the tongue epithelium. All animals developed clinical disease within two days and FMDV was reisolated from serum and saliva samples. In the second trial, another group of six cattle was exposed to FMDV RNA by instilling it on the tongue and spraying it into the nose. The animals were observed for 10 days after exposure. All animals remained clinically unremarkable and virus isolation as well as FMDV genome detection in serum and saliva were negative. No transfection reagent was used for any of the animal inoculations. In conclusion, cattle can be infected by injection with naked FMDV RNA, but not by non-invasive exposure to the RNA. Inactivated FMDV samples that contain full-length viral RNA carry only a negligible risk of infecting animals.

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BACKGROUND: RNA isolation from ossified bone is a difficult and time-consuming process which often results in poor recovery of RNA. The yield is limited and might not be suitable for gene quantification studies by real time PCR. METHODOLOGY: The present study demonstrates RNA extraction from rat femur utilizing the silica column along with the trizol reagent. Quality of RNA was assessed by agarose gel analysis and its suitability for real-time PCR analysis was determined by ß-actin Ct values. RESULTS: The RNA isolated using silica columns in conjugation with trizol reagent resulted in higher yield of RNA and purity (A260/280=2.04; yield =1545.73 µg/ml) compared to the trizol method alone (A260/280=1.85; yield =571.2 µg/ml). Ct value of ß actin obtained from RNA isolated by trizol method was higher than the Ct value obtained by trizol in conjugation with the column method (31.41 and 15.41 respectively). CONCLUSION: Combination of trizol along with silica column resulted in better quality and improved yield of RNA suitable for gene quantification by Real time PCR.

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Inactivation methods allow for hazard group 3 (HG3) pathogens to be disposed of and used safely in downstream experiments and assays to be carried out at lower containment levels. Commonly used viral inactivation methods include heat inactivation, fixation methods, ultraviolet (UV) light and detergent inactivation. Here we describe known methods used to inactivate SARS-CoV-2 for safe downstream biological assays.

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Research investigating the gut microbiome (GM) during a viral infection may necessitate inactivation of the fecal viral load. Here, we assess how common viral inactivation techniques affect 16S rRNA-based analysis of the gut microbiome. Five common viral inactivation methods were applied to cross-matched fecal samples from sixteen female CD-1 mice of the same GM background prior to fecal DNA extraction. The V4 region of the 16S rRNA gene was amplified and sequenced from extracted DNA. Treatment-dependent effects on DNA yield, genus-level taxonomic abundance, and alpha and beta diversity metrics were assessed. A sodium dodecyl sulfate (SDS)-based inactivation method and Holder pasteurization had no effect on measures of microbial richness, while two Buffer AVL-based inactivation methods resulted in a decrease in detected richness. SDS inactivation, Holder pasteurization, and the AVL-based inactivation methods had no effect on measures of alpha diversity within samples or beta diversity between samples. Fecal DNA extracted with TRIzol-treated samples failed to amplify and sequence, making it unsuitable for microbiome analysis. These results provide guidance in the 16S rRNA microbiome analysis of fecal samples requiring viral inactivation.

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Several studies have indicated the presence of microRNAs (miRNAs) within mitochondria although the origin, as well as the biological function, of these mitochondrially located miRNAs is largely unknown. The identification and significance of this subcellular localization is gaining increasing relevance to the pathogenesis of certain disease states. Here, we describe the isolation of highly purified mitochondria from rat liver by differential centrifugation, followed by RNAse A treatment to eliminate contaminating RNA. The coupled extraction of total RNA and protein is a more efficient design for allowing the downstream evaluation of miRNA and protein expression in mitochondria.

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Diagnostic testing is important for managing the 2019 novel coronavirus (SARS-CoV-2). We developed an optimized protocol for SARS-CoV-2 RNA extraction from the surface of the respiratory mucosa with nasopharyngeal swabs and compared the sensitivity of RNA extraction methods. RNA extraction was performed using three different procedures (TRIzol, QIAamp, VMT-TRIzol) from nine positive SARS-CoV-2 samples. SARS-CoV-2 was detected by real-time reverse transcriptase PCR (RT-PCR) using a detection kit for SARS-CoV-2 (Sun Yat-sen University). Compared to RT-PCR results, there were no discernible differences in detection rates when comparing the three different extraction procedures. On the basis of these results, the use of TRIzol as a transport medium and RNA extraction method for SARS-CoV-2 detection may be a helpful alternative for laboratories facing shortages of commercial testing kits.

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TRIzol is used for the extraction of RNA, DNA and proteins from tissues or cells. Here, we present a simple picking method to extract DNA from tissues using TRIzol. Spectrophotometric analysis showed that the 260/280 and 260/230 nm optical density ratio of the picking method's DNA is ideal and better than that obtained by the classic TRIzol method. Gel electrophoresis showed that there was no RNA contamination, and the DNA had not degraded. DNA extracted by the picking method had the same performance in restriction enzyme digestion and quantitative PCR as that obtained by the traditional method. Viral DNA in the infected tissue was also obtained. This modified method facilitates various molecular biology assays.

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TRIzol is a monophasic solution of phenol and guanidine isothiocyanate used for the extraction of RNA, DNA and proteins from tissues or cells. However, few studies have described its application to DNA extraction due to its time-consuming procedure. We present a TRIzol-modified method of extracting DNA from tissues using the TRIzol reagent and a silica column, which requires only one-third of the time required for the classic extraction procedure. Spectrophotometric analysis showed that the 260/280 and 260/230 nm optical density ratio of the DNA extracted using the TRIzol-modified method is ideal and equal to that obtained by the classic method and commercial DNAiso methods. The DNA extracted by the TRIzol-modified method had the same performance in a restriction enzyme digestion and quantitative PCR as that extracted using the classic method. Using the TRIzol-modified method saves time, simplifies the DNA extraction procedure, and facilitates various molecular biology assays.

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