BioTechniques

Efficient nucleic acid extraction and purification are crucial for cellular and molecular biology research, yet they pose challenges for large-scale clinical RNA sequencing and PCR assays. Here, we present BLADE-R, a magnetic bead-based protocol that simplifies the process by combining cellular lysis and nucleic acid binding into a single step, followed by a unique on-bead rinse for nuclease-free separation of genomic DNA and RNA. The Agilent TapeStation and RT-qPCR analyses show that RNA extracted from HEK293T cell line using BLADE-R outperforms the TRIzol protocol in terms of time and cost. RNA sequencing reveals no differences in sequence quality or gene count variance between samples processed with BLADE-R and those processed with TRIzol followed by RNA kit clean-up. Additionally, BLADE-R outperformed TRIzol in RNA extraction from frozen tissue and whole blood samples, as confirmed by RT-qPCR. Our protocol can be adapted to a 96-well plate format, enabling RNA purification of up to 96 human blood samples in less time than a single-sample traditional extraction. Using BLADE-R in this format, we confirmed minimal well-to-well contamination in RNA purification, cDNA synthesis, and PCR. Therefore, our novel BLADE-R protocol, suitable for both low and high-throughput formats, is effective even in limited-resource settings for preparing clinical samples for PCR and sequencing assays. Thus, our new BLADE-R technique works well even in low-resource environments to prepare clinical samples for PCR and sequencing experiments. It can be adapted for both low- and high-throughput formats.

Current methods for detecting, accurately quantifying, and profiling complex microbial communities based on the microbial 16S rRNA marker genes are limited by a number of factors, including inconsistent extraction of microbial nucleic acids, amplification interference from contaminants and host DNA, different coverage of PCR primers utilized for quantification and sequencing, and potentially biases in PCR amplification rates among microbial taxa during amplicon barcoding. Here, we describe a single-step method that enables the quantification of microbial 16S rRNA gene DNA copies with wide dynamic range and broad microbial diversity, and simultaneous amplicon barcoding for quantitative 16S rRNA gene amplicon profiling of microbiota. The method is suitable for a variety of sample types and is robust in samples with low microbial abundance, including samples containing high levels of host mammalian DNA, as is common in human clinical samples. We demonstrate that our modification to the Earth Microbiome Project (EMP) V4 16S rRNA gene primers expands their microbial coverage while dramatically reducing non-specific mammalian mitochondrial DNA amplification, thus achieving wide dynamic range in microbial quantification and broad coverage for capturing high microbial diversity in samples with or without high host DNA background. The approach relies only on broadly available hardware (real-time PCR instruments) and standard reagents utilized for conventional 16S rRNA gene amplicon library preparation both of which make it amenable for immediate and widespread adoption. Simultaneous 16S rRNA gene DNA copy quantification and amplicon barcoding for multiplexed next-generation sequencing from the same analyzed sample, performed in a combined workflow, reduces the amount of sample needed and reduces time and reagent costs. Additionally, we demonstrate that using our modified 16S rRNA gene primers in a digital PCR (dPCR) format enables precise and exact microbial quantification in samples with very high host DNA background levels without the need for quantification standards. Potential future applications of this approach include: (1) quantitative microbiome profiling in human and animal microbiome research; (2) detection of monoinfections and profiling of polymicrobial infections in tissues, stool, and bodily fluids in human and veterinary medicine; (3) environmental sample analyses (e.g., soil and water); and (4) broad-coverage detection of microbial food contamination in products high in mammalian DNA, such as meat products. We predict that utilization of this approach primarily for quantitative microbiome profiling will be invaluable to microbiome studies, which have historically been limited to analysis of relative abundances of microbes.

Ensuring COVID-19 testing remains accurate and reliable is of critical importance as the SARS-CoV-2 virus continues to evolve. Currently, a number of Omicron variants are dominating infection across the globe in including BQ.1 and XBB. Both variants and their sublineages (BQ.1* and XBB*) contain a 28,311 C/U mutation inherited from the original Omicron variant (BA.1). This mutation overlaps with a commonly used fluorescent probe for N gene detection in many Emergency Use Authorization (EUA) assays, as this target was originally established by the U.S. Centers for Disease Control and Prevention (CDC) in their EUA test for COVID-19 (2019-nCoV_N1). This C to U mutation was previously shown to have no impact on CDC N1 target detection. The rise of Omicron sublineages has increased the likelihood of additional point mutations occurring within the same assay target. A subpopulation of BQ.1* has an additional 28,312 C/U mutation within the CDC 2019_nCoV_N1 fluorescent probe in addition to the 28,311 C/U mutation. The double mutation could adversely affect the ability of diagnostic assays to detect the virus in patient samples and therefore it is important to verify the impacts of this additional mutation. Using in vitro transcribed (IVT) N gene RNA representing the wildtype (GenBank/GISAID ID MN908947.3) and Omicron BQ.1.1 variant (BQ.1, GISAID ID EPI_ISL_ 15155651), we evaluated the performance of two different amplification protocols, both of which include the CDC 2019-nCoV_N1 primer-probe set. Both assays successfully detected the mutant N gene sequence efficiently even at 10 copies of input, although the double mutation caused a 0.5[~]1 Cq delay on average when compared to the wild-type sequence. These data suggest that circulating BQ.1* lineage viruses with this double mutation likely have minimal impact on diagnostic assays that use the 2019-nCoV-N1 primer-probe.

Argonaute proteins are characterized by highly efficient guide DNA/RNA-directed binding to target nucleic acids with high fidelity. Employing this feature, we designed an argonaute-facilitated PCR platform by making use of an argonaute derived from Mesorhizobium japonicum (MejAgo) which does not carry nuclease activity and exposes the 3 end of guide DNA when binding. Each reaction cycle of the MejAgo-PCR platform consists of a denaturing step and a polymerase-mediated extension step, omitting the annealing step required by the traditional PCR. More importantly, MejAgo-PCR could significantly improve the sensitivity of PCR in template detection due to the argonaute-facilitated pairing between guide DNA/primer and template. Thus, an argonaute-facilitated PCR has the potential to be developed as an advanced PCR platform with higher sensitivity and efficiency.

Current biospecimen storage and preservation methods no longer meet the demands of basic research and clinical diagnostics. Biospecimen preservation methodology has not advanced to accommodate cutting edge molecular analysis technologies that target single cells and full-length transcripts. Traditional methods, such as flash freezing and formalin-fixed paraffin-embedding (FFPE), were designed to provide information on cell structure and spatial relationships in whole tissues. These methods, however, do not maintain the integrity of proteins and nucleic acids. In this proof-of-concept study we examine preservation mechanisms utilized in nature for survival during cold seasons or periods of drought. Plants, brine shrimp, and tardigrades rely on components such as disaccharides or intrinsically disordered proteins to maintain cellular structure and biological activity in the settings of these environmental stresses. Our study demonstrates that these reagents aid in mammalian cell preservation for at least one week when a sample is stored in solution at 4{degrees}C, or as a dried sample in a low humidity desiccator.

Determining RNA integrity is a critical quality assessment tool for gene expression studies where the experiments success is highly dependent on sample quality. Since its introduction in 1999, the gold standard in the scientific community has been the Agilent 2100 Bioanalyzers RNA Integrity Number (RIN) which uses a 1-10 value system with 1 being the most degraded to 10 being the most intact. In 2015, Agilent launched the 4200 Tapestations RIN equivalent and reported a strong correlation of r2 of 0.936 and median error < {+/-} 0.4 RIN units. To evaluate this claim, we compared the Agilent 4200 Tapestations RIN equivalent (RINe) and DV200 to the Agilent 2100 Bioanalyzers RIN for 183 parallel RNA samples. In our study, using RNA from a total of 183 human postmortem brain samples, we found that the RIN and RINe values only weakly correlate with an r2 of 0.393 and an average difference of 3.2 RIN units. DV200 also only weakly correlated with RIN (r2 of 0.182) and RINe (r2 of 0.347). Finally, when applying a cut-off value of 6.5 for both metrics, we found that 95.6% of samples passed with RIN, while only 23.5% passed with RINe. Our results suggest that even though RIN (Bioanalyzer) and RINe (Tapestation) use the same 1-10 value system, they should not be used interchangeably, and cut-off values should be calculated independently.

In situ hybridization (ISH) of RNA is a key method to visualize gene expression patterns in complex biological samples. The technique is indispensable for biological research related to e.g. development, disease, gene function, and the validation of novel cell types identified by single-cell sequencing methods. Especially in non-mammalian models lacking accessibility to a broad spectrum of antibodies, ISH remains a major research tool. Diverse available ISH protocols require different custom hybridization probe types, design, and/or proprietary signal detection chemistry. This makes it hard to navigate for a beginner and increases the research costs when multiple methods need to be applied. Here, we describe OneSABER - a unified open platform connecting commonly used canonical and recently developed single- and multiplex, colorimetric, and fluorescent ISH approaches. This platform uses a single type of ISH DNA probes adapted from the signal amplification by exchange reaction (SABER) method. We demonstrate applications of the proposed ISH framework in whole-mount samples of the regenerative flatworm Macrostomum lignano, advancing this animal as a powerful model for stem cell and regeneration research.

BackgroundPolyphosphates (polyP) and ATP are phosphate-containing metabolites present in prokaryotes and eukaryotes. PolyP has a wide variety of functions including phosphate and cation storage. ATP is a central metabolite in cellular bioenergetics and the phosphate providing substrate of polyP. In the green microalga Chlamydomonas reinhardtii, polyP synthesis is suggested to buffer ATP concentration, and the role of polyP in energetic metabolism requires further investigation. In this aim, relative quantification of both metabolites is needed. Because ATP and polyP half-lives differ greatly, harvesting generates biases in this relative quantification in current methods. For this reason, we present here a joint protocol optimised to compromise between maximal yield and specific constraints of both assays. MethodsThe optimised method quantifies ATP and polyP from the same C. reinhardtii cell extract after neutral phenol-chloroform extraction. Cells are directly pipetted from the culture to the phenol-chloroform- EDTA extraction mix. After a second chloroform extraction, ATP is quantified directly from the extract, while polyP measurement requires purification by ethanol precipitation. We used one-way analysis of variance or Kruskall-Wallis testing and appropriate post hoc testing to evaluate statistical effects in our results. ResultsWe show that he polyP/ATP ratio of the reference strain CC-4533 in exponential mixotrophic growth is around 65. While the optimised protocol performs as well as specific protocols for either ATP or polyP, the dispersion of the polyP/ATP ratio is twice better than for the separate metabolites. Direct sampling from the culture works better than centrifugation and filtration to maintain physiological conditions and high polyP yield. Using spiking with ATP and polyP, we show that ATP and longer chain polyP are fully recovered but not very short chain polyP. Finally, we show that the polyP chain length distribution extracted from CC-4533 is very broad, reaching up to several thousand P with a mean around 200 P. DiscussionOur protocol improves the precision of relative quantification of ATP and polyP by using neutral phenol-chloroform extraction and allows polyP/ATP ratio calculation from low-density samples and without normalisation. It can be applied to other microorganisms or cells, in a variety of physiological and stress conditions.

We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7x106 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.

The relative contributions of the conformation and primary structure of an epitope to overall antigen-antibody interaction (AAI) at denatured, native or formalin fixed (FF) state were compared using six randomly chosen commercial antibodies using Quantitative Dot Blot (QDB) method. AAIs at native and FF states were found ranged 1.3 [~] 10.2 and 0.5 [~] 45.4 folds, respectively, over those at denatured state in cellular and tissue lysates. Using two antibodies against different epitopes of PYGL protein, we showed that PYGL levels in several types of tissues and cell lines were highly correlated (r=0.99 from Pearson, p<0.0001, n=25) when measured with these two antibodies at native state. Yet, one antibody was found to be nonspecific with one type of these tissues using Western blot analysis. These observations suggested that the conformation of an epitope may serve as dominant contributor of overall AAI at native state in general, regardless of linear or conformational epitopes. In many cases, it would override nonspecific interactions formed at denatured state to challenge Western blot analysis as a validation tool for antibodies intended for immunohistochemistry (IHC) and ELISA.

Microbiota are a major component of agroecosystems. Root microbiota, which inhabit the inside and surface of plant roots, play a significant role in plant growth and health. As next-generation sequencing technology allows the capture of microbial profiles without culturing the microbes, profiling of plant microbiota has become a staple tool in plant science and agriculture. Here, we have developed a novel high-throughput method based on a two-step PCR amplification protocol, involving DNA extraction using magnetic beads and PCR purification using exonuclease, for 16S rRNA gene amplicon sequencing of plant root microbiota. This method reduces sample handling and captures microbial diversity comparable to that obtained by the standard method. We found that using a buffer with magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. In addition, we demonstrated that purification using exonuclease before the second PCR step enabled the capture of higher degrees of microbial diversity, thus allowing for the detection of minor bacteria compared with the purification using magnetic beads in this step. Our method offers a simple and high-throughput solution for maintaining the quality of plant root microbial community profiling.

Methods to visualize gene expression in the Drosophila central nervous system are important in fly neurogenetic studies. In this chapter, we describe a detailed protocol that sequentially combines in situ hybridization chain reaction (HCR) and immunostaining to detect mRNA and protein expression in whole-mount Drosophila larval and adult central nervous systems. We demonstrate the application of in situ HCR in comparisons of nervous system gene expression between Drosophila species, and in the validation of single-cell RNA-Seq results in the fly nervous system. Our protocol provides a simple, robust, multiplexable, and relatively affordable means to quantitatively visualize gene expression in the nervous system of flies, facilitating its general use in fly neurogenetic studies.

Bioluminescence is the production of visible light by living organisms through biochemical reactions in which specialized enzymes known as luciferases catalyze the oxidation of light-emitting substrates (luciferins), producing photons. In the ocean, bioluminescence is widespread among anthozoans (corals and sea anemones), yet the molecular basis of their light emission remains poorly defined. To date, molecular characterization within Anthozoa has largely focused on the sea pansy Renilla reniformis (Octocorallia: Renillidae), leaving anthozoan luciferase diversity underexplored. Here, we report the identification and biochemical characterization of a coelenterazine-dependent luciferase from the deepsea sea pen Anthoptilum murrayi (AnmLuc). Transcriptome analysis identified a transcript encoding a [~]34-kDa protein bearing motifs characteristic of coelenterazine-dependent luciferases. The coding sequence was cloned and the recombinant protein was expressed in Escherichia coli. The purified enzyme produced intense blue emission ({lambda}max {approx} 495 nm) in the presence of coelenterazine and displayed an activity optimum near 5 {degrees}C. These findings provide the first molecular characterization of a luciferase from a deep-sea anthozoan, expand the known diversity of coelenterazine-dependent luciferases in Cnidaria, and offer new insights into the mechanism and evolution of light emission in Anthozoa.

Highly multiplexed protein and RNA in situ concurrent detection on a single tissue section is highly desirable for both basic and applied biomedical research. CODEX is a new and powerful platform to visualize up to 60 protein biomarkers in situ and RNAscope in situ hybridization (RNAscope) is a novel RNA detection system with single-copy sensitivity and unprecedent specificity at a single cell level. Nevertheless, to our knowledge, the combination CODEX and RNAscope remained unreported until this study. Here we report a simple and reproducible combination of CODEX and RNAscope (Comb-CODEX-RNAscope). We also determined the cross-reactivities of CODEX anti-human antibodies to rhesus macaques, a widely used animal model of human disease.

We describe a cost-effective, highly sensitive, and quantitative method for in situ detection of single RNA molecules in tissue sections. This method, dubbed Yn situ, standing for Y-branched probe in situ hybridization, uses a single-strand DNA preamplifier with multiple initiation sites that trigger hybridization chain reaction (HCR) to detect polynucleotide. We characterized the performance of this method and compared it to other approaches in the postnatal mouse olfactory epithelia. We find that the Yn situ method, in conjunction with an improved fixation step, is sensitive enough to allow detection of single molecules using a single pair of probes targeting a short nucleotide sequence. A set of 5-probes can produce quantitative results with smaller puncta and higher signal-to-noise ratio than the 20-probe sets commonly required for HCR and RNA-Scope. We show that the high sensitivity and wide dynamic range allow quantification of genes expressed at different levels in the olfactory sensory neurons. We describe key steps of this method to enable broad utility by individual laboratories.

The preservation of ancient DNA in archaeological remains enables identification of past disease agents. However, pathogen DNA is typically highly diluted by host and environmental DNA, limiting detection. Here, we present a proof-of-concept study using in-solution hybridization capture to improve detectability of a pre-defined set of 12 pathogens. We validate the method by detecting Yersinia pestis, the plague agent, in six individuals from 17th and 18th century French plague cemeteries with minimal sequencing. Expanding our probe set to target biomarkers of virtually any pathogen of interest offers a powerful tool for tracking the prevalence of infectious diseases in ancient populations.

AbstractThe fungal pathogen Batrachochytrium salamandrivorans (Bsal) is one of two species (the other, B. dendrobatidis/Bd) that cause amphibian chytridiomycosis, an emerging infectious disease that has been indicated in the declines of hundreds of amphibian species worldwide. While Bd has been near-globally distributed for well over a century, Bsal is a more recently emerged pathogen, having been identified just over a decade ago with current impacts localized to salamandrids in parts of Europe. However, because there is concern that Bsal will cause widespread declines if introduced to naive regions - such as the Americas where the greatest diversity of salamandrids exist - it is imperative that widespread testing and monitoring strategies be implemented to mitigate the spread of Bsal. As standard diagnostic approaches tend to be expensive, time-consuming, or require specialized instrumentation and training, we have developed a simplified, rapid, CRISPR-based approach for Bsal-DNA identification and provide suggestions for its future application.

Current RNA purification methods widely use silica-based columns that allow quick isolation of high-quality and good quantities of RNA. However, the major limitations include high cost, the requirement of different kits for small RNA isolation, genomic DNA contamination, and not being flexible. Here, we used the in-house RNA isolation reagent (RIR) for cell lysis, followed by RNA precipitation using isopropanol. RNA isolated using the in-house RIR resulted in a similar quantity and quality compared to the commercial TRIzol. Furthermore, the commercial RNA isolation kits with silica-based columns recommend genomic DNA digestion during or after RNA purification, adding time and cost to RNA purification. Here, we developed an optimized in-house protocol for isolating high-quality RNA free of genomic DNA contamination using magnetic silica beads without needing DNase digestion. Additionally, our method purifies total RNA along with the small RNA fraction, including miRNAs, which usually require a separate kit for extraction. Additionally, the RNA prepared with our method was equally suitable for mRNA and miRNA expression analysis using RT-qPCR. Together, the in-house method of RNA isolation using the magnetic silica beads has exhibited comparable or better total RNA extraction compared to commercial kits at a fraction of the cost and across various cells and tissues.

Here we provide technical data for amplifying the complete genome of SARS-CoV-2 from clinical samples using only seventeen pairs of primers. We demonstrate that the [C]V2000bp primer panel successfully produces genomes when used with the residual total RNA extracts from positive clinical samples following diagnostic RT-PCRs (with Ct in the range from 13 to 20). The library preparation method reported here includes genome amplification of ~1750-2000 bp fragments followed by ultrasonic fragmentation combined with the introduction of Illumina compatible adapters. Using the SCV2000bp panel, 25 complete SARS-CoV-2 virus genome sequences were sequenced from clinical samples of COVID-19 patients from Moscow obtained in late March - early April.

TBK1 is a serine-threonine kinase that has been linked to a number of diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Reproducible research on TBK1 has been hampered by the lack of well characterized antibodies. In this study, we characterized 11 commercial antibodies for immunoblot, immunofluorescence and immunoprecipitation, using a knock-out cell line as the control. For each application, we identified several potent and selective antibodies that will facilitate studies on TBK1.