Search

turboLysis is a novel mechanical cell lysis device that utilizes small beads to efficiently lyse tough cells like Mycobacterium, Saccharomyces, and Arabidopsis. We compared turboLysis to bead beating using the BeadBug 6 for several concentrations of Mycobacterium tuberculosis roughly correlated to the bacterial load commonly seen in patient samples. turboLysis performed similarly to the BeadBug at low bacterial concentrations and outperformed it at high concentrations above 2x105 CFU/ml (p

Gene expression studies are widely used in medical, biological, and pharmaceutical research. Obtaining high-quality RNA from tissues is a prerequisite for high-quality data that should accurately represent gene expression levels in-vivo. The main source of technical bias, which could affect the results from transcriptomic studies, is variation in RNA quality. In this regard, tissue preparation is critical: different disruption techniques can affect RNA quality, influencing further applications. Mechanical disaggregation is a common, inexpensive, and simple method to obtain a high cell yield, demonstrated to efficiently disrupt the extracellular matrix and release single cells. However, its efficacy is operator-dependent, leading to poorly reproducible results. A fast, reproducible, and standardized technique could undoubtedly overcome this problem, avoiding wasting time and resources. In this study, our goal was to evaluate the impact of two mechanical tissue disruption techniques on the purity and quality of RNA extracted from fresh lung biopsies. The samples were processed in parallel using manual mechanical disaggregation or an automated mechanical device. The results showed that samples processed with the automated device had a higher integrity compared to those processed manually with a median Fragmentation Index of 0.86 and 0.71 respectively. This difference is statistically significant (p = 0.0084). Overall, our results indicated that the use of automatic mechanical disaggregation could undoubtedly help to overcome the technical biases related to fresh tissues processing. [ABSTRACT FROM AUTHOR]

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 β‐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. The conventional Trizol method was modified by adding PVPP for grinding, as well as using Chloroform, CTAB, and CH3COONa instead of a phenol, chloroform, and isoamyl alcohol mixed solution. The present investigation assesses and enhances the methods employed for extracting RNA from E. senticosus. The modified Trizol method 2, which has facilitated the successful isolation of premium RNA from E. senticosus leaves, stands as a convenient and cost‐effective extraction protocol and can promote research in plant molecular biology. [ABSTRACT FROM AUTHOR]

Since early 2024, a multistate outbreak of highly pathogenic avian influenza H5N1 has been affecting dairy cattle in the USA. The influenza viral RNA concentrations in milk make it an ideal matrix for surveillance purposes. However, viral RNA detection in multi-component fluids such as milk can be complex, and optimization of influenza detection methods is thus required. Raw bulk tank milk and mastitis milk samples were artificially contaminated with an avian influenza strain and subjected to five extraction methods. HCoV-229E and synthetic RNA were included as exogenous internal process controls. Given the high viral load usually observed in individual raw milk samples, four out of five tested methods would enable influenza detection in milk with normal texture, over a time window of at least 2 weeks post-onset of clinical signs. Nevertheless, sample dilution 1:3 in molecular transport medium prior to RNA extraction provided the best results for dilution of inhibitory substances and a good recovery rate of influenza RNA, that reached 12.5 ± 1.2% and 10.4 ± 3.8% in two independent experiments in bulk milk and 11.2 ± 3.6% and 10.0 ± 2.9% on two cohorts of mastitis milk samples. We have also shown compatibility of an influenza RT-qPCR system with synthetic RNA detection for simultaneous validation of the RNA extraction and RT-qPCR processes. [ABSTRACT FROM AUTHOR]

Balamuthia mandrillaris is a free-living amoeba (FLA) that causes granulomatous amebic encephalitis (GAE) and skin lesions. Transcriptomic analysis is a powerful tool used to study B. mandrillaris pathogenic infections. However, preliminary tests of RNA extraction showed poor results, so it has become essential to standardize a protocol for high-quality RNA. The present study evaluated 11 RNA extraction protocols based on three commercial kits by making modifications to the temperature and centrifugation times, and by combining kits. Four protocols, namely Q3 (based on QIAGEN RNeasy Mini Kit, with modifications in temperature and centrifugation times), T1 (Invitrogen TRIzol Reagent), T2 (combination of TRIzol and QIAGEN modified protocols) and T3 (combination of TRIzol and PROMEGA SV Total RNA Isolation protocols), presented RNA with good integrity and purity, except for the T1 protocol, which obtained an A260/230 value below the acceptable threshold. High RNA integrity (RIN) values were obtained with the Q3 (9.8), T2 (9.2), and T3 (8.9) protocols, while the T1 protocol obtained a lower RIN value (7.1). The Q3, T2, and T3 protocols obtained high-quality RNA from B. mandrillaris based on the criteria of integrity, purity, and concentration, where the implemented modifications and combinations raised the quality; thus, their use is recommended to obtain accurate results when performing transcriptomic analysis.

Background: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. Methods: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. Results: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. Conclusions: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis. [ABSTRACT FROM AUTHOR]

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus's diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning. [ABSTRACT FROM AUTHOR]

Balamuthia mandrillaris is a free-living amoeba (FLA) that causes granulomatous amebic encephalitis (GAE) and skin lesions. Transcriptomic analysis is a powerful tool used to study B. mandrillaris pathogenic infections. However, preliminary tests of RNA extraction showed poor results, so it has become essential to standardize a protocol for high-quality RNA. The present study evaluated 11 RNA extraction protocols based on three commercial kits by making modifications to the temperature and centrifugation times, and by combining kits. Four protocols, namely Q3 (based on QIAGEN RNeasy Mini Kit, with modifications in temperature and centrifugation times), T1 (Invitrogen TRIzol Reagent), T2 (combination of TRIzol and QIAGEN modified protocols) and T3 (combination of TRIzol and PROMEGA SV Total RNA Isolation protocols), presented RNA with good integrity and purity, except for the T1 protocol, which obtained an A260/230 value below the acceptable threshold. High RNA integrity (RIN) values were obtained with the Q3 (9.8), T2 (9.2), and T3 (8.9) protocols, while the T1 protocol obtained a lower RIN value (7.1). The Q3, T2, and T3 protocols obtained high-quality RNA from B. mandrillaris based on the criteria of integrity, purity, and concentration, where the implemented modifications and combinations raised the quality; thus, their use is recommended to obtain accurate results when performing transcriptomic analysis. [ABSTRACT FROM AUTHOR]

turboLysis is a novel mechanical cell lysis device that utilizes small beads to efficiently lyse tough cells like Mycobacterium, Saccharomyces, and Arabidopsis. We compared turboLysis to bead beating using the BeadBug 6 for several concentrations of Mycobacterium tuberculosis roughly correlated to the bacterial load commonly seen in patient samples. turboLysis performed similarly to the BeadBug at low bacterial concentrations and outperformed it at high concentrations above 2x105 CFU/ml (p

Abstract Background High-quality RNA extraction from woody plants is difficult because of the presence of polysaccharides and polyphenolics that bind or co-precipitate with the RNA. The CTAB (cetyl trimethylammonium bromide) based method is widely used for the isolation of nucleic acids from polysaccharide-rich plants. Despite the widespread use of the CTAB method, it is necessary to adapt it to particular plant species, tissues and organs. Here we described a simple and generalized method for RNA isolation from mature leaf tissues of several economically important woody (17) and herbaceous plants (2) rich in secondary metabolites. High yields were achieved from small amount (up to 50 mg) of plant material. Two main modifications were applied to the basic protocol: an increase in β-mercaptoethanol concentration (to 10%v/v) and the use of an effective DNase treatment. As opposed to similar studies, we tried to describe a more detailed protocol for isolating RNA, including the exact quantity and concentration of the reagents were used. Results Our modified CTAB method is proved to be efficient in extracting the total RNA from a broad range of woody and herbaceous species. The RNA yield was ranged from 2.37 to 91.33 µg/µl. The A260:A280 and A260:A230 absorbance ratios were measured from 1.77 to 2.13 and from 1.81 to 2.22. The RIN value (RNA Integrity Number) of the samples fell between 7.1 and 8.1, which indicated that a small degree of RNA degradation occurred during extraction. The presence of a single peak in the melt curve analyses and low standard errors of the Ct values of replicated measurements indicated the specificity of the primers to bind to the cDNA. Conclusions Our RNA isolation method, with fine-tuned and detailed instructions, can produce high quality RNA from a small amount of starting plant material that is suitable for use in downstream transcriptional analyses. The use of an increased concentration of the reducing agent β-mercaptoethanol in the extraction buffer, as well as the application of DNaseI-treatment resulted in a method suitable for a wide range of plants without the need of further optimalization, especially in Rhus typhina (Staghorn sumac), for which molecular-genetic studies have not yet been sufficiently explored.

Abstract Background Blood transcriptomic analysis is widely used to provide a detailed picture of a physiological state with potential outcomes for applications in diagnostics and monitoring of the immune response to vaccines. However, multi-species transcriptomic analysis is still a challenge from a technological point of view and a standardized workflow is urgently needed to allow interspecies comparisons. Results Here, we propose a single and complete total RNA-Seq workflow to generate reliable transcriptomic data from blood samples from humans and from animals typically used in preclinical models. Blood samples from a maximum of six individuals and four different species (rabbit, non-human primate, mouse and human) were extracted and sequenced in triplicates. The workflow was evaluated using different wet-lab and dry-lab criteria, including RNA quality and quantity, the library molarity, the number of raw sequencing reads, the Phred-score quality, the GC content, the performance of ribosomal-RNA and globin depletion, the presence of residual DNA, the strandness, the percentage of coding genes, the number of genes expressed, and the presence of saturation plateau in rarefaction curves. We identified key criteria and their associated thresholds to be achieved for validating the transcriptomic workflow. In this study, we also generated an automated analysis of the transcriptomic data that streamlines the validation of the dataset generated. Conclusions Our study has developed an end-to-end workflow that should improve the standardization and the inter-species comparison in blood transcriptomics studies. In the context of vaccines and drug development, RNA sequencing data from preclinical models can be directly compared with clinical data and used to identify potential biomarkers of value to monitor safety and efficacy.

Peripheral blood is the most practical tissue for human immune system gene expression profiling because it is easily accessible, whereas the site of primary infection in certain diseases may not be easily accessible. Due to the ex vivo instability of RNA transcripts, a key challenge in the gene expression analysis of blood samples is the rapid sample handling and stabilization of the mRNA by adding an RNA preservative (PAXgeneTM Blood RNA Tubes, TempusTM Blood RNA tubes, RNAlater Stabilization Reagent, RNAgard® Blood Tubes). BioMole (Turin, Italy) has developed a novel blood stabilizer, called RNApro, in which RNA is stabilized during phlebotomy and sample storage. In this study, RNApro performance intended as RNA yield, integrity, and stability was evaluated. Our results show that blood samples stored at −80 °C and re-extracted after 7 years show no differences in terms of quantity, quality, and amplificability. The samples in the RNAlater stabilization solution can be stored at room temperature for up to one week or at 4 °C for up to one month. Similar results can also be observed for PAXgene tubes, Tempus tubes, and RNAgard tubes. In agreement with these data, the RNApro stabilization solution preserves the RNA from degradation for up to 1 month at 4 °C and 1 week at room temperature. RNApro can be stored indifferently at −80, −20, 4 °C, or room temperature for up to 2 months after, and then could be stored at −80 °C for up to seven years. In summary, our study is the first to analyze the performance of an RNA stabilizer called RNApro. We can conclude that several studies have shown significant differences in gene expression analysis when the sample was preserved in different RNA stabilizers. Therefore, it is desirable to standardize the method of nucleic acid conservation when comparing data from transcriptomic analyses. [ABSTRACT FROM AUTHOR]

Human noroviruses (HuNoVs) are among the main causes of acute gastroenteritis worldwide. HuNoVs can survive for several days up to weeks at room temperature in the environment, on food, and on food handling and processing surfaces. As a result, this could lead to viral spread through the ingestion of food in contact with contaminated surfaces. The development of stable surface materials with antiviral activity might be useful to reduce viral outbreaks. Metal-based compounds, including photoactivated titanium nanoparticles (TiO2 NPs), are known for their antiviral activity. In this study, we tested the impact of 2000 µg/mL TiO2 NPs, with or without UV activation, on HuNoV GII and murine norovirus. Their recovery rates were reduced by 99.6%. We also evaluated a new TiO2 NP-coating process on a polystyrene surface. This process provided a homogenous coated surface with TiO2 NPs ranging between 5 nm and 15 nm. Without photoactivation, this TiO2 NP-coated polystyrene surface reduced the recovery rates of intact HuNoV GII by more than 94%. When a capsid integrity treatment with PtCl4 or a longer reverse transcription polymerase chain detection approach was used to evaluate virus integrity following contact with the TiO2 NP-coated polystyrene, the HuNoV GII recovery yield reduction varied between 97 and 100%. These results support the hypothesis that TiO2 NP-coated surfaces have the potential to prevent viral transmission associated with contaminated food surfaces. [ABSTRACT FROM AUTHOR]

Background: High-quality RNA extraction from woody plants is difficult because of the presence of polysaccharides and polyphenolics that bind or co-precipitate with the RNA. The CTAB (cetyl trimethylammonium bromide) based method is widely used for the isolation of nucleic acids from polysaccharide-rich plants. Despite the widespread use of the CTAB method, it is necessary to adapt it to particular plant species, tissues and organs. Here we described a simple and generalized method for RNA isolation from mature leaf tissues of several economically important woody (17) and herbaceous plants (2) rich in secondary metabolites. High yields were achieved from small amount (up to 50 mg) of plant material. Two main modifications were applied to the basic protocol: an increase in β-mercaptoethanol concentration (to 10%v/v) and the use of an effective DNase treatment. As opposed to similar studies, we tried to describe a more detailed protocol for isolating RNA, including the exact quantity and concentration of the reagents were used. Results: Our modified CTAB method is proved to be efficient in extracting the total RNA from a broad range of woody and herbaceous species. The RNA yield was ranged from 2.37 to 91.33 µg/µl. The A260:A280 and A260:A230 absorbance ratios were measured from 1.77 to 2.13 and from 1.81 to 2.22. The RIN value (RNA Integrity Number) of the samples fell between 7.1 and 8.1, which indicated that a small degree of RNA degradation occurred during extraction. The presence of a single peak in the melt curve analyses and low standard errors of the Ct values of replicated measurements indicated the specificity of the primers to bind to the cDNA. Conclusions: Our RNA isolation method, with fine-tuned and detailed instructions, can produce high quality RNA from a small amount of starting plant material that is suitable for use in downstream transcriptional analyses. The use of an increased concentration of the reducing agent β-mercaptoethanol in the extraction buffer, as well as the application of DNaseI-treatment resulted in a method suitable for a wide range of plants without the need of further optimalization, especially in Rhus typhina (Staghorn sumac), for which molecular-genetic studies have not yet been sufficiently explored. [ABSTRACT FROM AUTHOR]

RNA extraction from formalin‐fixed paraffin‐embedded tissues (FFPE) presents a notable challenge. Extracting RNA from FFPE tissues has been challenging due to RNA degradation and chemical bonding between RNA and proteins during the fixation process. This results in reduced concentration and quality of the extracted RNA from FFPE tissues, impacting subsequent downstream analyses. In this study, we present an optimized approach involves replacing the tissue temperature‐digestion step with tissue crushing and homogenization. RNA extraction was performed on 100 FFPE tissue blocks obtained from the archive of Pathology Department of Shiraz University of Medical Sciences. After deparafinization of the tissues, RNA was extracted using this modified method, and the results compared to those of a standard extraction procedure and commercial kits used for FFPE tissue. Then gel electrophoresis and Real time PCR were done to check the quality and integrity of the extracted RNA. The results show that the concentration of RNA obtained from this method was significantly (P value <0.0001) higher than the other methods. In conclusion, we have successfully introduced a modified method that enables the extraction of high‐quality and intact RNA from FFPE tissue. This method stands as a reliable option for molecular biology studies necessitating precise RNA extraction. [ABSTRACT FROM AUTHOR]

In 2020, there were numerous cases in Kazakhstan with clinical symptoms of COVID-19 but negative PCR results in nasopharyngeal and oropharyngeal swabs. The diagnosis was confirmed clinically and by CT scans (computed tomography). The problem with such negative PCR results for SARS-CoV-2 infection confirmation still exists and indicates the need to confirm the diagnosis in the bronchoalveolar lavage in such cases. There is also a lack of information about confirmation of SARS-CoV-2 infection in deceased patients. In this study, various tissue materials, including lungs, bronchi, and trachea, were examined from eight patients who died, presumably from SARS-CoV-2 infection, between 2020 and 2022. Naso/oropharyngeal swabs taken from these patients in hospitals tested PCR negative for SARS-CoV-2. This study presents a modified RNA isolation method based on a comparison of the most used methods for RNA isolation in laboratories: QIAamp Viral RNA Mini Kit and TRIzol-based method. This modified nucleic acid extraction protocol can be used to confirm SARS-CoV-2 infection by RT-qPCR in the tissues of deceased patients in disputed cases. RT-qPCR with RNA of SARS-CoV-2 re-extracted with such method from post-mortem tissues that were stored at -80 °C for more than 32 months still demonstrated high-yielding positive results. [ABSTRACT FROM AUTHOR]

Background: Blood transcriptomic analysis is widely used to provide a detailed picture of a physiological state with potential outcomes for applications in diagnostics and monitoring of the immune response to vaccines. However, multi-species transcriptomic analysis is still a challenge from a technological point of view and a standardized workflow is urgently needed to allow interspecies comparisons. Results: Here, we propose a single and complete total RNA-Seq workflow to generate reliable transcriptomic data from blood samples from humans and from animals typically used in preclinical models. Blood samples from a maximum of six individuals and four different species (rabbit, non-human primate, mouse and human) were extracted and sequenced in triplicates. The workflow was evaluated using different wet-lab and dry-lab criteria, including RNA quality and quantity, the library molarity, the number of raw sequencing reads, the Phred-score quality, the GC content, the performance of ribosomal-RNA and globin depletion, the presence of residual DNA, the strandness, the percentage of coding genes, the number of genes expressed, and the presence of saturation plateau in rarefaction curves. We identified key criteria and their associated thresholds to be achieved for validating the transcriptomic workflow. In this study, we also generated an automated analysis of the transcriptomic data that streamlines the validation of the dataset generated. Conclusions: Our study has developed an end-to-end workflow that should improve the standardization and the inter-species comparison in blood transcriptomics studies. In the context of vaccines and drug development, RNA sequencing data from preclinical models can be directly compared with clinical data and used to identify potential biomarkers of value to monitor safety and efficacy. [ABSTRACT FROM AUTHOR]

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at −80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies. [ABSTRACT FROM AUTHOR]

Background and Aims Transcriptome sequencing is a cost-effective approach that allows researchers to study a broad range of questions. However, to preserve RNA for transcriptome sequencing, tissue is often kept in special conditions, such as immediate ultracold freezing. Here, we demonstrate that RNA can be obtained from 6-month-old, field-collected samples stored in silica gel at room temperature. Using these transcriptomes, we explore the evolutionary relationships of the genus Pitcairnia (Bromeliaceae) in the Dominican Republic and infer barriers to gene flow. Methods We extracted RNA from silica-dried leaf tissue from 19 Pitcairnia individuals collected across the Dominican Republic. We used a series of macro- and micro-evolutionary approaches to examine the relationships and patterns of gene flow among individuals. Key Results We produced high-quality transcriptomes from silica-dried material and demonstrated that evolutionary relationships on the island match geography more closely than species delimitation methods. A population genetic examination indicates that a combination of ecological and geographical features presents barriers to gene flow in Pitcairnia. Conclusions Transcriptomes can be obtained from silica-preserved tissue. The genetic diversity among Pitcairnia populations does not warrant classification as separate species, but the Dominican Republic contains several barriers to gene flow, notably the Cordillera Central mountain range. [ABSTRACT FROM AUTHOR]

Introduction: Human saliva was used to develop non-invasive liquid biopsy biomarkers to establish saliva as an alternate to blood and plasma in translational research. The present study focused on understanding the impact of sample storage conditions on the extraction of RNA from saliva and the RNA yield, to be applied in clinical diagnosis. In this study, genes related to asthma were used to test the method developed.Methods: Salivary RNA was extracted from three subjects using the Qiazol® based method and quantified by both spectrophotometric (NanoDrop) and fluorometric (Qubit®) methods. RNA integrity was measured using a bioanalyzer. Quantitative PCR was used to monitor the impact of storage conditions on the expression of housekeeping genes: GAPDH and β-actin, and the asthma related genes: POSTN and FBN2. In addition, an independent cohort of 38 asthmatics and 10 healthy controls were used to validate the expression of POSTN and FBN2 as mRNA salivary biomarkers.Results: Approximately 2 µg of total RNA was obtained from the saliva stored at 40°C without any preservative for 2 weeks showing consistent gene expression with RNA stored at room temperature (RT) for 48 h with RNAlater. Although saliva stored with RNAlater showed a substantial increase in the yield (110 to 234 ng/μL), a similar Cq (15.6 ± 1.4) for the 18s rRNA gene from saliva without preservative showed that the RNA was stable enough. Gene expression analysis from the degraded RNA can be performed by designing the assay using a smaller fragment size spanning a single exon as described below in the case of the POSTN and FBN2 genes in the asthma cohort.Conclusion: This study showed that samples stored at room temperature up to a temperature of 40°C without any preservative for 2 weeks yielded relatively stable RNA. The methodology developed can be employed to transport samples from the point of collection to the laboratory, under non-stringent storage conditions enabling the execution of gene expression studies in a cost effective and efficient manner.

Since the end of 2019, COVID-19 pandemic caused by the novel SARS-CoV-2 has become a serious problem for the world. Accurate and rapid techniques in testing and tracing are needed to control the virus spreading. Molecular diagnostics through gene amplification techniques, especially PCR, still become the gold standard for SARS-CoV-2 detection, which requires the first step of RNA extraction and purification. The limitations of commercial RNA extraction-purification kits during the pandemic caused a big problem in testing and tracing, especially for developing countries. A simple RNA extraction-purification kit based on magnetic-silica (MAGSi) beads and non-guanidine in-house buffers for RNA virus extraction-purification has been developed. Two types of MAGSi beads with different magnetic nanoparticles (MNPs) content were synthesized through a modified Stöber’s method using the sonication technique. The PCR result shows that both the MAGSi beads and the buffer can be used as a kit for RNA extraction-purification, tested for SARS-CoV-2 and Canine Distemper Virus. Further study shows that MAGSi-1 has better RNA extraction ability, and a higher concentration of RNA has been extracted. This is likely because of the smaller particle size distribution (50–1,500 nm distribution) and higher magnetization (20.2 emu/g) of MAGSi-1 compared to MAGSi-2 with 100–1,700 nm size distribution and 14.2 emu/g magnetization.

Since early 2024, a multistate outbreak of highly pathogenic avian influenza H5N1 has been affecting dairy cattle in the USA. The influenza viral RNA concentrations in milk make it an ideal matrix for surveillance purposes. However, viral RNA detection in multi-component fluids such as milk can be complex, and optimization of influenza detection methods is thus required. Raw bulk tank milk and mastitis milk samples were artificially contaminated with an avian influenza strain and subjected to five extraction methods. HCoV-229E and synthetic RNA were included as exogenous internal process controls. Given the high viral load usually observed in individual raw milk samples, four out of five tested methods would enable influenza detection in milk with normal texture, over a time window of at least 2 weeks post-onset of clinical signs. Nevertheless, sample dilution 1:3 in molecular transport medium prior to RNA extraction provided the best results for dilution of inhibitory substances and a good recovery rate of influenza RNA, that reached 12.5 ± 1.2% and 10.4 ± 3.8% in two independent experiments in bulk milk and 11.2 ± 3.6% and 10.0 ± 2.9% on two cohorts of mastitis milk samples. We have also shown compatibility of an influenza RT-qPCR system with synthetic RNA detection for simultaneous validation of the RNA extraction and RT-qPCR processes.

In order to obtain RNA from wine grape leaves with high purity and good quality for fluorescence real-time quantitative analysis, and to provide technical support for subsequent studies on the regulation of transcription levels using wine grape leaf RNA as materials and have certain application value, in this study, classic wine grape Cabernet Sauvignon leaves were used as the experimental materials. Effects of four methods, including resteaming phenol, Trizol, improved cetyltrimethyl ammonium bromide(CTAB), and LiCl(based on improved SDS), on RNA extraction from wine grape leaves were compared and analyzed. The results showed that the concentration of RNA extracted by resteaming phenol was low, the integrity was poor, and there was partial degradation. In addition, the RNA extracted by Trizol method had some DNA contamination. In the modified CTAB method, the extraction concentration was high, but the protein and DNA contamination was serious. In the RNA swim lane extracted by LiCl method, the brightness of the 28S band was twice of that of the 18S band. The band has no dragging phenomenon, and the extracted RNA integrity was good, but there was still DNA contaminationn and a small amount of protein contamination. Further, on the basis of LiCl method, sodium acetate, digestion by DNAase, and phenol extraction method were used for optimization. The results showed that the addition of sodium acetate did not improve the quality of RNA. However, total RNA from grape leaves with high purity, good integrity, and no protein and DNA contamination (173.611 µg/mL, A260/280= 1.803) was extracted by extracting chloroform and water-saturated phenol(1:1) twice, chloroform once, chloroform and water-saturated phenol(1∶1) once, and digestion by DNAase. The results of real-time fluorescence quantitative PCR further verified that the RNA extracted by this method obtained good amplification curve and melting curve after reverse transcription, and could be used to quantitatively analyze related genes. [ABSTRACT FROM AUTHOR]

The golden-headed lion tamarin (Leontopithecus chrysomelas) is an endangered primate endemic to the Atlantic Forest. Conservation efforts for the species involve applying reproductive biotechniques to preserve genetic resources and ensure the management of populations in both ex situ and in situ conditions. This study aims to initiate investigations into seminal and molecular factors influencing the reproductive potential of sexually mature males. Semen was collected using the penile vibrostimulation technique, and seminal parameters were assessed in two groups: the 'Old' group (average age 11.6 years; n=6) and the 'Young' group (average age 4.8 years; n=6). ANOVA results indicated age-related influences on plasma membrane integrity (p=0.049), acrosomal integrity (p=0.009), and DAB IV (p=0.026) for both groups. Linear regression revealed significant correlations between seminal parameters and age (plasma membrane integrity (p=0.021), acrosomal integrity (p=0.05), and DAB III (p=0.024)), alongside animal weight (plasma membrane integrity (p=0.010), acrosomal integrity (p=0.009), DAB III (p=0.33), and DAB IV (p=0.066)). In an effort to advance reproductive techniques and sperm selection, a protocol utilizing a discontinuous Percoll gradient was employed. Despite its effectiveness in isolating gametes, there were no significant gains in the reevaluated parameters post-selection, necessitating adjustments in the methodology. While semen cryopreservation is common in wild species, challenges arise due to seminal coagulum in many neotropical primate ejaculates, hindering gamete use in reproductive procedures. Given the precious nature of and the considerable effort involved in collecting semen from these animals, it would be desirable to maximize the sample's utility. The liquid fraction could be applied in reproductive biotechniques, while the spermatozoa contained in the clot could be utilized as a non-invasive approach for molecular evaluation of these gametes. This study established a protocol for RNA extraction from sperm retained in the seminal coagulum, highlighting its genetic richness often discarded post-processing. In summary, our study emphasizes the importance of early cryopreservation of semen to safeguard the reproductive potential of L. chrysomelas. Additionally, we propose further exploration of RNA quantity in gametes as a non-invasive tool for inferring male fertility, given the pivotal role of sperm RNA transcripts in regulating the activation of the female gamete and gene expression during early embryo development.

Human noroviruses (HuNoVs) are among the main causes of acute gastroenteritis worldwide. HuNoVs can survive for several days up to weeks at room temperature in the environment, on food, and on food handling and processing surfaces. As a result, this could lead to viral spread through the ingestion of food in contact with contaminated surfaces. The development of stable surface materials with antiviral activity might be useful to reduce viral outbreaks. Metal-based compounds, including photoactivated titanium nanoparticles (TiO2 NPs), are known for their antiviral activity. In this study, we tested the impact of 2000 µg/mL TiO2 NPs, with or without UV activation, on HuNoV GII and murine norovirus. Their recovery rates were reduced by 99.6%. We also evaluated a new TiO2 NP-coating process on a polystyrene surface. This process provided a homogenous coated surface with TiO2 NPs ranging between 5 nm and 15 nm. Without photoactivation, this TiO2 NP-coated polystyrene surface reduced the recovery rates of intact HuNoV GII by more than 94%. When a capsid integrity treatment with PtCl4 or a longer reverse transcription polymerase chain detection approach was used to evaluate virus integrity following contact with the TiO2 NP-coated polystyrene, the HuNoV GII recovery yield reduction varied between 97 and 100%. These results support the hypothesis that TiO2 NP-coated surfaces have the potential to prevent viral transmission associated with contaminated food surfaces.

Peripheral blood is the most practical tissue for human immune system gene expression profiling because it is easily accessible, whereas the site of primary infection in certain diseases may not be easily accessible. Due to the ex vivo instability of RNA transcripts, a key challenge in the gene expression analysis of blood samples is the rapid sample handling and stabilization of the mRNA by adding an RNA preservative (PAXgeneTM Blood RNA Tubes, TempusTM Blood RNA tubes, RNAlater Stabilization Reagent, RNAgard® Blood Tubes). BioMole (Turin, Italy) has developed a novel blood stabilizer, called RNApro, in which RNA is stabilized during phlebotomy and sample storage. In this study, RNApro performance intended as RNA yield, integrity, and stability was evaluated. Our results show that blood samples stored at −80 °C and re-extracted after 7 years show no differences in terms of quantity, quality, and amplificability. The samples in the RNAlater stabilization solution can be stored at room temperature for up to one week or at 4 °C for up to one month. Similar results can also be observed for PAXgene tubes, Tempus tubes, and RNAgard tubes. In agreement with these data, the RNApro stabilization solution preserves the RNA from degradation for up to 1 month at 4 °C and 1 week at room temperature. RNApro can be stored indifferently at −80, −20, 4 °C, or room temperature for up to 2 months after, and then could be stored at −80 °C for up to seven years. In summary, our study is the first to analyze the performance of an RNA stabilizer called RNApro. We can conclude that several studies have shown significant differences in gene expression analysis when the sample was preserved in different RNA stabilizers. Therefore, it is desirable to standardize the method of nucleic acid conservation when comparing data from transcriptomic analyses.

[Display omitted] • TiO 2 NTs@Fe 3 O 4 NPs-assisted open-channel device for viral RNA extraction. • Direct viral RNA detection via simple fluorescence status evaluation. • Method validated by 145 SARS-CoV-2 samples against PCR-based method. • Nanomaterial-assisted device meets the criteria of point-of-care testing. Most routine viral RNA detection assays available today rely on RNA amplification steps. The sensitivity of such methods comes at the expense of time, cost, and equipment availability. Given the constraints during the COVID-19 pandemic, such as the limited capacity of RNA amplification instruments and the global shortage of isolation kits, there has been a demand for amplification-free assays which should meet POCT criteria: affordable, sensitive, specific, user-friendly, rapid, and equipment-free. This study aims to design and validate a microanalytical system for direct viral RNA detection which meets these criteria. The presented RNA detection assay consists of four consecutive steps: immunocapturing viral particles, viral particles lysis, TiO 2 NTs@Fe 3 O 4 NPs-assisted RNA extraction, and finally viral RNA detection. The last three steps occur within a microfluidic device with open channels (OC-MFD). The OC-MFD is manufactured from polycarbonate film by unique mechanical replication techniques in a roll-to-roll setup. This method has been experimentally validated to offer the advantages of low-cost and extremely rapid production (1 mil. pieces of OC-MFDs per hour). Two permanent magnets situated under the application/extraction zones enabled to fix magnetically active particles/nanotubes. At first, viral particles are quantitatively captured from the entire sample volume using immunomagnetic particles. The lysed viral particles are transferred to the application zone of OC-MFD, followed by RNA extraction using magnetic TiO 2 nanotubes decorated with Fe 3 O 4 nanoparticles (TiO 2 NTs@Fe 3 O 4 NPs). The effectivity of the nanomaterial to attract viral RNA molecules was evaluated against a standard RT-qPCR-based laboratory test. Equivalence tests were performed with 143 positive and negative SARS-CoV-2 samples. Spearman rank correlation coefficients demonstrated high agreement for both measured biomarker genes (the E gene equaled 0.76, the RdRp gene equaled 0.73 , the p-value < 0.001). To fulfill POCT criteria, viral RNA captured directly on the surface of TiO 2 NTs@Fe 3 O 4 NPs and fixed by permanent magnet in the extraction zone of OC-MFD was visualized in situ using SYBR Green II fluorescent dye. This strategy allows to achieve a sensitivity of 0.3 ng of RNA per µL−1. Additionally, this test can be easily adapted for direct detection of any other RNA virus simply by choosing a suitable immunosorbent with the corresponding antibody specificity. Besides, the most significant advantages of this microanalytical system for direct viral RNA detection and outlook for future applications are also discussed and summarized. [ABSTRACT FROM AUTHOR]

The extraction of high-quality RNA from cotton (Gossypium spp.) is challenging because of the presence of high polyphenolics, polysaccharides, quinones, and other secondary metabolites. A high-throughput RNA extraction protocol is a prerequisite. This Triton-X-100-based RNA extraction method utilizes Polyvinyl pyrrolidone polymer (PVPP) treatment which efficiently removes phenolics, and the application of Lithium chloride (LiCl) has been found that successfully precipitated the high-quality RNA from cotton tissue. Cytoplasmic male sterility (CMS) is a maternally inherited trait associated with specific mitochondrial genome rearrangements or mutations. The suitability of RNA extracted from Cotton CMS lines was assessed. cDNA was synthesized from RNA and assayed for mitochondrial genes (cox3, nad3, nad9) associated with male sterility. This paper discuss the advantages and limitation of this protocol over existing protocol for RNA extraction for polyphenolics-rich plant tissue. [Display omitted] • Efficient protocol with a minimum amount of plant material is required. • Extraction protocol is equally useful for plants having diverse lipids/fatty acids, polysaccharides, and polyphenols. • High throughput protocol is simple, economic, less time-consuming, efficient and applicable to cytoplasmic genome analysis. [ABSTRACT FROM AUTHOR]

RT-PCR is the gold standard for diagnosis of COVID-19. All RT-PCR kits are based on RNA extraction from the clinical sample. There was a sudden increase in demand of these kits, both RNA extraction and COVID-19 RT-PCR kits during the pandemic. This sudden spurt in global demand created a situation of shortage of consumables, especially the RNA extraction kits. Hence, this study was carried out to evaluate and compare COVID-19 RT-PCR without RNA extraction step using buffer R3. Sensitivity, specificity and accuracy of RT-PCR kit without RNA extraction were 89.16 %, 100% and 89.6% respectively. This approach saved more than 50 % time compared to the RT-PCR kit with RNA extraction approach allowing enhanced daily sample processing capability. RT-PCR kit without RNA extraction help in managing a greater number of samples, reduces cost and turnaround time. [ABSTRACT FROM AUTHOR]

Globally, porcine rotavirus is a leading cause of gastroenteritis in nursing and post-weaning piglets, as well as adult pigs. Between February 2015 and June 2016, 156 fecal samples were collected from pigs in the Northeastern part of Accra, Ghana, and screened for Group A rotavirus using the Proflow TM Kit. Here, we describe different extraction methods that were employed to recover high-quality RNA for downstream analysis, with emphasis on a novel hybrid extraction method. The hybrid approach with a kit and manual extraction method led to a 10-fold greater RNA yield versus the kit-based method alone. The new extraction method gave an average purity ratio (A 260 /A 280 ) of 1.8, which was also significantly higher than that obtained solely from the manual or kit-based extraction methods. Our novel hybrid approach will be useful in the extraction of rotavirus from animal fecal samples, thus improving the yield of RNA for downstream analysis. © 2024 Wiley Periodicals LLC. Basic Protocol: Hybrid 2: A double lysis method for RNA extraction from animal stool samples Support Protocol 1: The GenElute extraction method Support Protocol 2: Hybrid 1 extraction method., (© 2024 Wiley Periodicals LLC.)

Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed-specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time-consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size-exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high-quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Robust polysome extraction for seeds Basic Protocol 2: Rapid fraction total RNA extraction., (© 2024 Wiley Periodicals LLC.)

Wastewater-based epidemiology has allowed tracking the magnitude and distribution of SARS-CoV-2 in communities, allowing public health officials to prepare for impending outbreaks. While many factors influence recovery of SARS-CoV-2 from wastewater, proper extraction, concentration, and purification of RNA are key steps to ensure accurate detection of viral particles. The aim of this study was to compare the efficiency of four commonly used RNA extraction methods for detection of the SARS-CoV-2 RNA genome in sewage samples artificially inoculated with the virus, in order to identify a protocol that improves viral recovery. These methods included CTAB-based, TRIzol-based, and guanidinium thiocyanate (GTC)-based extraction procedures coupled with silica spin column-based purification, and an automated extraction/purification protocol using paramagnetic particles. Following RNA extraction, virus recovery rates were compared using RT-qPCR-based detection. The CTAB-based approach yielded the highest recovery rates and was the only method to consistently demonstrate stable virus recovery percentages regardless of the specific physicochemical characteristics of the samples tested. The TRIzol method proved to be the second most effective, yielding significantly higher recovery rates compared to both the GTC-based and the automated extraction methods. These results suggest that the CTAB-based approach could be a useful tool for the recovery of viral RNA from complex wastewater matrices. [Display omitted] • Extraction methods were compared for the recovery of SARS-CoV-2 RNA from sewage. • CTAB-PVP-column and TRIzol-column methods showed the highest recovery rates. • CTAB-PVP-column was the most efficient when sewage presented high turbidity grades. • CTAB-PVP-column recovery rates were independent of the matrix´s characteristics analyzed. • This procedure could be useful for the recovery of viral RNA from complex matrices. [ABSTRACT FROM AUTHOR]

turboLysis is a novel mechanical cell lysis device that utilizes small beads to efficiently lyse tough cells like Mycobacterium, Saccharomyces, and Arabidopsis. We compared turboLysis to bead beating using the BeadBug 6 for several concentrations of Mycobacterium tuberculosis roughly correlated to the bacterial load commonly seen in patient samples. turboLysis performed similarly to the BeadBug at low bacterial concentrations and outperformed it at high concentrations above 2x10 5 CFU/ml (p < 0.005). Thus, turboLysis offers good cell lytic performance in a small form factor at a low cost., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

This study investigated the influence of relative humidity (RH) on the efficiency of SARS-CoV-2 RNA extraction using the Nextractor automated system. Experiments employing clinical samples demonstrated satisfactory sensitivity and reproducibility for RNA extraction at low humidity (below 50% RH). Conversely, extractions at high humidity (above 70% RH) resulted in complete failure of reverse transcription-polymerase chain reaction assays, with neither SARS-CoV-2 RNA nor the human RNase P gene (internal control) detected. Analysis suggested that residual ethanol, incompletely evaporating due to high humidity, acted as a potent polymerase chain reaction inhibitor in these samples. These findings highlighted the importance of maintaining optimal laboratory humidity (<50% RH) for reliable SARS-CoV-2 RNA extraction using the Nextractor system. Furthermore, laboratories should implement strategies such as regular humidity monitoring, staff training on humidity's impact, and system validation under specific humidity conditions to ensure accurate molecular diagnostic workflows for COVID-19 testing., Competing Interests: The authors declare no conflict of interest., (© 2024 The Journal of Biological Methods, All rights reserved.)

Background: Early diagnosis of pancreatic ductal adenocarcinoma (PDAC) has been a longstanding challenge. The prognosis of patients with PDAC depends on the stage at diagnosis. It is necessary to identify biomarkers for the detection and differentiation of pancreatic tumors and optimize PDAC sample preparation procedures for DNA and RNA analysis. Most molecular studies are done using paraffin-embedded blocks; however, the integrity of DNA and RNA is often compromised in this format. Moreover, RNA isolated from human pancreatic tissue samples is generally of low quality, in part, because of the high concentration of endogenous pancreatic RNAse activity present., Aim: To assess the potential of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to obtain specimens from pancreatic neoplasms for subsequent RNA molecular profiling, including next-generation sequencing (NGS)., Methods: Thirty-four EUS-FNA samples were included in this study: PDAC ( n = 15), chronic pancreatitis ( n = 5), pancreatic cysts ( n = 14), mucinous cysts (mucinous cystic neoplasia/intraductal papillary mucinous neoplasia) n = 7, serous cystic neoplasms n = 5, and pseudocysts n = 2. Cyst material consisted of cyst fluid and cyst wall samples obtained by through-the-needle biopsy (TTNB). Samples were stored at -80 °C until analysis. RNA purity (A260/230, A260/280 ratios), concentration, and integrity (RIN) were assessed. Real-time polymerase chain reaction was conducted on all samples, and small RNA libraries were prepared from solid mass samples., Results: RNA was successfully extracted from 29/34 (85%) EUS-FNA samples: 100% pancreatic adenocarcinoma samples, 100% chronic pancreatitis samples, 70% pancreatic fluid cyst samples, and 50% TTNB samples. The relative expression of GAPDH and HPRT were obtained for all successfully extracted RNA samples ( n = 29) including low-quality RNA specimens. Low concentration and nonoptimal RIN values (no less than 3) of RNA extracted from EUS-FNA samples did not prevent NGS library preparation. The suitability of cyst fluid samples for RNA profiling varied. The quality of RNA extracted from mucinous cyst fluid had a median RIN of 7.7 (5.0-8.2), which was compatible with that from solid neoplasms [6.2 (0-7.8)], whereas the quality of the RNA extracted from all fluids of serous cystic neoplasms and TTNB samples had a RIN of 0., Conclusion: The results demonstrate the high potential of EUS-FNA material for RNA profiling of various pancreatic lesions, including low-quality RNA specimens., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Introduction: Human saliva was used to develop non-invasive liquid biopsy biomarkers to establish saliva as an alternate to blood and plasma in translational research. The present study focused on understanding the impact of sample storage conditions on the extraction of RNA from saliva and the RNA yield, to be applied in clinical diagnosis. In this study, genes related to asthma were used to test the method developed. Methods: Salivary RNA was extracted from three subjects using the Qiazol ® based method and quantified by both spectrophotometric (NanoDrop) and fluorometric (Qubit ® ) methods. RNA integrity was measured using a bioanalyzer. Quantitative PCR was used to monitor the impact of storage conditions on the expression of housekeeping genes: GAPDH and β-actin, and the asthma related genes: POSTN and FBN2 . In addition, an independent cohort of 38 asthmatics and 10 healthy controls were used to validate the expression of POSTN and FBN2 as mRNA salivary biomarkers. Results: Approximately 2 µg of total RNA was obtained from the saliva stored at 40°C without any preservative for 2 weeks showing consistent gene expression with RNA stored at room temperature (RT) for 48 h with RNA later . Although saliva stored with RNA later showed a substantial increase in the yield (110 to 234 ng/μL), a similar Cq (15.6 ± 1.4) for the 18s rRNA gene from saliva without preservative showed that the RNA was stable enough. Gene expression analysis from the degraded RNA can be performed by designing the assay using a smaller fragment size spanning a single exon as described below in the case of the POSTN and FBN2 genes in the asthma cohort. Conclusion: This study showed that samples stored at room temperature up to a temperature of 40°C without any preservative for 2 weeks yielded relatively stable RNA. The methodology developed can be employed to transport samples from the point of collection to the laboratory, under non-stringent storage conditions enabling the execution of gene expression studies in a cost effective and efficient manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bhamidimarri, Fuentes, Salameh, Mahboub and Hamoudi.)

The COVID-19 pandemic brought to light the impact of a widespread disease on various aspects of human relationships, communities, and economies. One notable consequence was the increased demand for diagnostic kits, laboratory reagents, and personal health equipment. This surge in testing capacity worldwide led to shortages in the supply of essential items, including RNA extraction kits, which are crucial for detecting COVID-19 infections. To address this scarcity, researchers have proposed alternative and cost-effective strategies for RNA extraction, utilizing both chemical and physical solutions and extraction-free methods. These approaches aim to alleviate the challenges associated with the overwhelming number of tests being conducted in laboratories. The purpose of this review is intends to provide a comprehensive summary of the various kit-free RNA extraction methods available for COVID-19 diagnosis during the pandemic. [Display omitted] • Using alternative process to bypass RNA extraction stage during an emergency situation such as the covid-19 pandemic. • Extraction free process such as using PK treatment, high temperature show acceptable results compare to standard process through real time pcr. • Different alternative RNA preparation process can reduce cost and increase testing speed. [ABSTRACT FROM AUTHOR]

Serum microRNAs (miRNAs) are considered useful as non-invasive biomarkers for different diseases. However, the optimal method for extracting RNAs from serum is currently unknown. In the present study, several RNA extraction kits were used to examine the optimal kit. RNAs were extracted from the serum of 8-week-old C57BL/6NJcl male mice following the protocol of each RNA extraction kit. The yield of the extracted RNA samples was calculated, and an Agilent Bioanalyzer was used to assess the electrophoretic patterns. An Agilent mouse miRNA microarray was utilized to confirm the expression patterns of the extracted RNA samples. The results revealed significant differences in RNA yields from the miRNeasy Serum/Plasma Advanced kit and mirVana™ PARIS™ RNA and Native Protein Purification Kit compared with almost all other samples. Further, two peaks were determined in the miRNeasy Serum/Plasma Advanced kit using a small RNAs kit of Agilent Bioanalyzer, including one at 20-40 nucleotides (nt) and another at ~40-100 nt, whereas the other reagents had a single peak. This revealed that the extracted RNAs may differ in composition based on the RNA extraction method. Some types of miRNAs were only detected with certain RNA extraction reagents. This suggested that different RNA extraction reagents may cause differences in the types of miRNAs detected. On the other hand, the miRNAs commonly expressed by the three RNA extraction reagents are highly correlated in expression levels. [ABSTRACT FROM AUTHOR]

Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcriptional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post-meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced fertility poses to the obtaining of experimental animals., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Post-translationally modified peptides are now recognized as important regulators of plant stress responses. We recently identified the sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in dark-grown and in light-stressed Arabidopsis seedlings. The function of CLEL6 depends on proteolytic processing by subtilisin-like serine proteinase SBT6.1, and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST), and CLEL6 signaling relies on the ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptor family. In this chapter, we describe in detail how to quantify peptide-regulated and stress-induced anthocyanin biosynthesis. We include protocols for peptide treatment of Arabidopsis seedlings and growth under different stress conditions, for the extraction and quantification of anthocyanins, and for the expression analysis of anthocyanin biosynthetic genes., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Octocorals are the most prolific source of terpenoids in the marine environment, with more than 4000 different compounds known from the phylum to date. However, the biochemical and genetic origin of their production remained elusive until recent studies showed that octocorals encode genes responsible for the biosynthesis of terpenoids in their own chromosomal DNA rather than from microbial symbionts as originally proposed. The identified coral genes include those encoding a new group of class I terpene cyclases (TCs) clustered among other candidate classes of tailoring enzymes. Phylogenetic analyses established octocoral TCs as a monophyletic clade, distinct from TCs of plants, bacteria, and other organisms. The newly discovered group of TCs appears to be ubiquitous in octocorals and is evolutionarily ancient. Given the recent discovery of octocoral terpenoid biochemistry and only limited genomic data presently available, there is substantial potential for discovering new biosynthetic pathways from octocorals for terpene production. The following chapter outlines practical experimental procedures for octocoral DNA and RNA extraction, genome and transcriptome assembly and mining, TC cloning and gene expression, protein purification, and in vitro analyses., (Copyright © 2024. Published by Elsevier Inc.)

Primary cell culture is an invaluable method frequently used to overcome challenges associated with in vivo experiments. In zebrafish research, in vivo live imaging experiments are routine owing to the high optical transparency of embryos, and, as a result, primary cell culture has been less utilized. However, the approach still boasts powerful advantages, emphasizing the importance of sophisticated zebrafish cell culture protocols. Here, we present an enhanced protocol for the generation of primary cell cultures by dissociation of 24 hpf zebrafish embryos. We include a novel cell culture medium recipe specifically favoring neuronal growth and survival, enabling relatively long-term culture. We outline primary zebrafish neuronal culture on glass coverslips, as well as in transwell inserts which allow isolation of neurite tissue for experiments such as investigating subcellular transcriptomes., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)