Modified Spin Column-Based RNA Extraction Methods of Staphylococcus aureus using PureLink® RNA Mini Kit and Basic Laboratory Instrument

RNA extraction is an important process before gene expression assessment at the transcriptomic level. RNA is a sensitive material to environmental factors such as temperature and contaminants, so the RNA extraction process generally requires sophisticated and expensive laboratory instruments. In this study, we extract RNA from Staphylococcus aureus bacteria using the PureLink® RNA Mini Kit. The instruments used in this study are basic instruments such as a hand homogenizer and non-thermal centrifuge. The results of RNA extraction were visualized using agarose gel electrophoresis. These results indicate that bacterial RNA extraction can be performed using the PureLink® RNA Mini Kit even with inexpensive basic laboratory instruments.

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.

Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

Nucleic acids, RNA among them, are widely used in biomedicine. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher. In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. These data contribute to facilitate researchers in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure to guarantee both quality standards and its reproducibility.

Saliva molecular testing bypassing RNA extraction is suitable for monitoring and diagnosing SARS-CoV-2 infection in children

IMPORTANCE Adults are being vaccinated against SARS-CoV-2 worldwide, but the longitudinal protection of these vaccines is uncertain, given the ongoing appearance of SARS-CoV-2 variants. Children are susceptible to infection, and some studies reported that they actively transmit the virus even when asymptomatic, thus affecting the community. Methods to easily test infected children and track the virus they carry are in demand. OBJECTIVE To determine if saliva is an effective sample for detecting SARS-CoV-2 RNA and antibodies in children aged 10 years and under, and associate viral RNA levels to infectivity. DESIGN, SETTING, AND PARTICIPANTS In this cross-sectional study, saliva SARS-CoV-2 RT-qPCR tests, with and without RNA extraction, were validated in 49 hospitalized adults. The test was then applied to 85 children, aged 10 years and under, admitted to the hospital regardless of COVID-19 symptomatology. Amongst 85 children, 29 (63.0%) presented at least one COVID-19 symptom, 46 (54.1%) were positive for SARS-CoV-2 infection, 28 (32.9%) were under the age of 1 and the mean (SD) age was 3.8 (3.4) years. Saliva samples were collected up to 48 h after a positive test by nasopharyngeal (NP) swab-RT-qPCR. EXPOSURE Infection by SARS-COV-2 in adults up to 8 days post-symptom onset. Children admitted to hospital for any reason and therefore with unclear onset of SARS-CoV-2 infection. MAIN OUTCOMES AND MEASURES Saliva RT-qPCR up to CT<37 accurately identifies SARS-CoV-2 infected children, with viral infectivity in tissue culture restricted to CT<26. RESULTS In adults, the accuracy of the saliva SARS-CoV-2 RT-qPCR test was 98.0% (95% confidence intervals [CI]: 89.3%-100%) as compared to NP-RT-qPCR. In children, the sensitivity, specificity, and accuracy of saliva-RT-qPCR tests compared to NP swab-RT-qPCR were, respectively, 84.8% (71.8%-92.4%), 100% (91.0%-100%), and 91.8% (84.0%-96.6%) with RNA extraction and 81.8% (68.0%-90.5%), 100% (91.0%-100%), and 90.4% (82.1%-95.0%) without RNA extraction. The threshold for rescuing infectious particles from saliva was CT<26. There were significant IgM positive responses to the spike protein and its receptor-binding domain (RBD) among children positive for SARS-CoV-2 by NP swab and negative by saliva compared to other groups, indicating late infection onset (>7-10 days). CONCLUSIONS AND RELEVANCE Saliva-molecular testing is suitable in children aged 10 years and under, including infants aged <1 year, even bypassing RNA extraction methods. Importantly, the detected viral RNA levels were significantly above the infectivity threshold in several samples. Further investigation is required to understand how SARS-CoV-2 RNA levels correlate with viral transmission.

Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods

Advances in high-throughput sequencing technologies have enabled extensive studies of freshwater biofilms and significant breakthroughs in biofilm meta-omics. To date, however, no standardized protocols have been developed for the effective isolation of RNA from freshwater benthic biofilms. In this study, we compared column-based kit RNA extraction with five RNAzol-based extractions, differentiated by various protocol modifications. The RNA products were then evaluated to determine their integrity, purity and yield and were subjected to meta-transcriptomic sequencing and analysis. Significant discrepancies in the relative abundance of active communities and structures of eukaryotic, bacterial, archaebacterial, and viral communities were observed as direct outcomes of the tested RNA extraction methods. The column isolation-based group was characterized by the highest relative abundance of Archaea and Eukaryota, while the organic isolation-based groups commonly had the highest relative abundances of Prokaryota (bacteria). Kit extraction methods provided the best outcomes in terms of high-quality RNA yield and integrity. However, these methods were deemed questionable for studies of active bacterial communities and may contribute a significant degree of bias to the interpretation of downstream meta-transcriptomic analyses.