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Caenorhabditis elegans is a well-established laboratory animal model and has been widely used in biological research. However, it is still a challenge to obtain a good amount of quality RNA from a limited number of C. elegans for gene expression studies. To address this issue, the present study has compared different conditions to preserve C. elegans for RNA extraction after the failure of an initial effort to use RNAlater-preserved worms for RNA extraction. The effects of different concentrations of proteinase K, different worm life stages, and different worm numbers on RNA extraction were also investigated. The best results were achieved under the following conditions: 1) adult worms that were either freshly prepared or quickly frozen in liquid nitrogen followed by storage at −80 °C; 2) disruption of C. elegans with proteinase K (1 mg/mL) in a lysis buffer (65 °C for 10 min) prior to extraction with Trizol agent. This method can provide a stable, rapid, and effective means to extract RNA from C. elegans with variable worm numbers from 20 to 200. • RNAlater was inappropriate for preserving C. elegans for effective RNA extraction. • Proteinase K was verified for lysing a limited number of C. elegans for RNA extraction. Method name: Inappropriateness of RNAlater to preserve C. elegans for RNA extraction, Keywords: Caenorhabditis elegans, RNA extraction, RNAlater, Storage conditions, Proteinase K

The SARS-CoV-2 pandemic has caused unpredictable mortality and economic losses globally. With no approved drug for the treatment, the accurate diagnosis of COVID-19 becomes essential. RNA based test takes several hours and require extensive human intervention for RNA extraction and RT-PCR, but it is preferred over the antibody-based detection as the latter does not detect the early stage infections. The RT-PCR being a gold standard of COVID-19 diagnosis offers highly standardized detection of the SARS-CoV-2 RNA, still vulnerable for false-negative diagnosis due to absence of infected cells in the sample or inaccurate RNA extraction. Hence there is a need to develop alternative protocols and methods for the accurate COVID-19 diagnosis. Here we propose two additional steps in RT-PCR based COVID-19 diagnosis to minimize false-negative detection. The first step involves collection of four samples from an individual. Each sample should be collected from nasopharyngeal and oropharyngeal regions on day 01, mixed together followed by RNA extraction and then repeating the same exercise on day 03. The RNA extracted on day 01 and day 03 must be pooled together to be used in the RT-PCR. Second, we propose the inclusion of the control marker genes specific to nasal goblet cell, type-II pneumocyte and absorptive enterocytes to ensure the specificity of the RNA source. Overall, these additional steps in the proposed method would increase the chances of SARS-CoV-2 detection in the infected population and would limit the false-negative diagnosis of COVID-19 and hence the spread of this disease.•RT-PCR based COVID-19 diagnosis is vulnerable to the false-negative results due to inaccurate sample isolation or RNA extraction.•RNA pool of multiple samples from an individual improves the chances of detection of SARS-CoV-2 by RT-PCR.•Inclusion of specific marker genes would ensure the right RNA source from the desired cell., Graphical abstract Image, graphical abstract

Graphical abstract, Transcriptomic analysis of single cells has been increasingly in demand in recent years, thanks to technological and methodological advances as well as growing recognition of the importance of individuals in biological systems. However, the majority of these studies have been performed in mammalian cells, due to their ease of lysis and high RNA content. No single cell transcriptomic analysis has yet been described in microbial spores, even though it is known that heterogeneity at the phenotype level exists among individual spores. Transcriptomic analysis of single spores is challenging, in part due to the physically robust nature of the spore wall. This precludes the use of methods commonly used for mammalian cells. Here, we describe a simple method for extraction and amplification of transcripts from single fungal conidia (asexual spores), and its application in single-cell transcriptomics studies. The method can also be used for studies of small numbers of fungal conidia, which may be necessary in the case of limited sample availability, low-abundance transcripts or interest in small subpopulations of conidia. • The method allows detection of transcripts from single conidia of Aspergillus niger • The method allows detection of genomic DNA from single conidia of Aspergillus niger

Noncoding RNAs (ncRNAs) comprise a class of versatile transcripts that are highly involved in the regulation of a wide range of biological processes. Functional long ncRNAs (> 200 nts in length) often adopt secondary structures that arise co-transcriptionally. To maintain the secondary structure elements as well as preparation homogeneity of such transcripts, native-like conditions should be maintained throughout the in vitro synthesis, purification and chemical tagging processes. In this optimized protocol, we describe a simple method for obtaining homogenous samples followed by chemically tagging the 3′ termini of natively-purified structured ncRNA domains that are longer than 200 nts. This protocol replaces traditional hazardous radioactive labeling with fluorescence tagging, and eliminates laborious and time consuming RNA purification and concentration steps and replaces them with straightforward recovery of RNA through centrifugal filtration, preserving the homogeneity and mono-dispersion of the preparations. The protocol provides:•An integrative, simple and straightforward approach for synthesis, purification and labeling of structured ncRNAs whilst maintaining their secondary structure intact.•Replacing hazardous, laborious and time-consuming radioactive labeling of RNA with much simpler fluorescence tagging, thereby facilitating potential downstream applications such as electrophoretic mobility shift assay (EMSA).•A versatile protocol that could be applicable to a wide-range of chemical tags and in principle could be used to label DNA or RNA., Graphical abstract Image, graphical abstract

Graphical abstract, Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis. • Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity. • Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity. • In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.

Graphical abstract, RNA and DNA extraction is a requirement for the study of gene expression and has an increasingly important role in genetic studies of all fleshy fruits. RNA and DNA extraction is difficult in kiwifruit due to the significant amount of polysaccharides and polyphenols compounds. So far, no commercial kit has been developed specifically for high-quality RNA and DNA extraction in kiwifruit and the common protocols for RNA extraction have poor yields. This study developed a new protocol for high quality RNA extraction in Actinidia deliciosa. According to the results, the average yield of RNA extraction of fruit and leaf of A. deliciosa was ∼2180.7 ng/μl (∼545.175 μg/g FW) and ∼3424.9 ng/μl (∼856.225 μg/g FW), respectively with A260/A280 between 1.95 to 2.07 and A260/A230 higher than 2 indicating high RNA purity. While the averages yield of RNA extraction using previous methods from kiwifruit and leaf was 23 μg/g FW and 527 μg/g FW, respectively. Also, the average yields of genomic DNA from kiwifruit ranged from 52 to 98 ng/μl with A260/A230 between 0.60 to 1.64 and A260/A280 between 1.40 to 1.48. To our knowledge, this is the first report of a highly efficient and rapid method of RNA and DNA extraction in kiwifruit which can be used for a broad spectrum of the all fleshy fruits.

Graphical abstract, Zebrafish is a powerful animal model used to study vertebrate embryogenesis, organ development and diseases (Gut et al., 2017) [1]. The usefulness of the model was established as a result of various large forward genetic screens identifying mutants in almost every organ or cell type (Driever et al., 1996; Haffter et al., 1996) [[2], [3]]. More recently, the advent of genome editing methodologies, including TALENs (Sander et al., 2011) [4] and the CRISPR/Cas9 technology (Hwang et al., 2013) [5], led to an increase in the production of zebrafish mutants. A number of these mutations are homozygous lethal at the embryonic or larval stages preventing the generation of homozygous mutant zebrafish lines. Here, we present a method allowing both genotyping and phenotype analyses of mutant zebrafish larvae from heterozygous zebrafish incrosses. The procedure is based on the genotyping of the larval tail after transection, whereas phenotypic studies are performed on the anterior part of the zebrafish larvae. • The method includes (i) a protocol for genotyping, (ii) protocols for paraffin embedding and histological analyses, (iii) protocols for protein and histone extraction and characterization by Western blot, (iv) protocols for RNA extraction and characterization by RT-PCR, and (v) protocols to study caudal spinal cord regeneration. • The technique is optimized in order to be applied on single zebrafish embryos and larvae.

Microbially-mediated hydrocarbon degradation is well documented. However, how these microbial processes occur in complex subsurface petroleum impacted systems remains unclear, and this knowledge is needed to guide technologies to enhance microbial degradation effectively. Analysis of RNA derived from soils impacted by petroleum liquids would allow for analysis of active microbial communities, and a deeper understanding of the dynamic biochemistry occurring during site remediation. However, RNA analysis in soils impacted with petroleum liquids is challenging due to: (A) RNA being inherently unstable, and (B) petroleum impacted soils containing problematic levels of polymerase chain reaction (PCR) inhibitors that must be removed to yield high-purity RNA for downstream analysis. A previously published soil wash pretreatment step and a commercially available DNA extraction kit protocol were combined and modified to be able to purify RNA from soils containing petroleum liquids.•A key modification involved reformulation of the pretreatment solution via replacing water as the diluent with a commercially-available RNA preservation solution.•Methods were developed and demonstrated using cryogenically preserved soils from three former petroleum refineries. Results showed the new soil washing approach had no adverse effects on RNA recovery but did improve RNA quality, by PCR inhibitor removal, which in turn allows for characterization of active microbial communities present in petroleum impacted soils.•In summary, our method for extracting RNA from petroleum-impacted soils provides a promising new tool for resolving metabolic processes at sites as they progress toward restoration via natural and/or engineered remediation., Graphical abstract Image, graphical abstract

A multitude of challenges is faced during RNA extraction from human visceral adipose tissue (VAT) due to its atypical nature and a dearth of existing literature. Our study provides a convenient and inexpensive manual method using TRIzol reagent for the reproducible recovery of intact RNA from sparse human VAT samples. Fifty-two (52) samples were grouped and tested for the effect of different factors viz. initial VAT amount, TRIzol volume per unit tissue mass, residual fat following homogenisation and first centrifugation, an additional chloroform wash, and an additional ethanol wash on the extraction process. We found that increasing initial tissue mass and decreasing TRIzol volume simultaneously improved RNA yield and purity. A fat layer removal step and additional ethanol wash further propel the A260/280 and A260/230 to their desired values. Our modifications in the isolation protocol were combined and tested through reverse transcriptase quantitative PCR, which yielded consistent results, upholding our optimisation., Graphical abstract Image, graphical abstract

Graphical abstract, Caenorhabditis elegans is a well-established laboratory animal model and has been widely used in biological research. However, it is still a challenge to obtain a good amount of quality RNA from a limited number of C. elegans for gene expression studies. To address this issue, the present study has compared different conditions to preserve C. elegans for RNA extraction after the failure of an initial effort to use RNAlater-preserved worms for RNA extraction. The effects of different concentrations of proteinase K, different worm life stages, and different worm numbers on RNA extraction were also investigated. The best results were achieved under the following conditions: 1) adult worms that were either freshly prepared or quickly frozen in liquid nitrogen followed by storage at −80 °C; 2) disruption of C. elegans with proteinase K (1 mg/mL) in a lysis buffer (65 °C for 10 min) prior to extraction with Trizol agent. This method can provide a stable, rapid, and effective means to extract RNA from C. elegans with variable worm numbers from 20 to 200. • RNAlater was inappropriate for preserving C. elegans for effective RNA extraction. • Proteinase K was verified for lysing a limited number of C. elegans for RNA extraction.

Graphical abstract, Traditional RNA extraction methods rely on the use of hazardous chemicals such as phenol, chloroform, guanidinium thiocyanate to disrupt cells and inactivate RNAse simultaneously. RNA isolation from Caenorhabditis elegans presents another challenge due to its tough cuticle, therefore several repeated freeze–thaw cycles may be needed to disrupt the cuticle before the cell contents are released. In addition, a large number of animals are required for successful RNA isolation. To overcome these issues, we have developed a simple and efficient method using proteinase K and a brief heat treatment to release RNA of quality suitable for quantitative PCR analysis.The benefits of the method are: • Faster and safer compared to conventional RNA extraction methods • Released RNA can be used directly for cDNA synthesis without purification • As little as a single worm is sufficient

Graphical abstract, RNA isolation from human or animal cartilage tissue is necessary when performing mechanical or biotribological applications. Despite no influence on the cells and no alterations in gene expression patterns, enzymatic digestion of tissues should be avoided as it’s known that the expression of collagen 2 can be effected (Hayman et al., 2006 [1]). After mechanical or biotribological tests alternative options with an immediate disruption of the tissue should be contemplated. To obtain RNA, different tissue homogenization and disruption methods are available on the market (Yu et al., 2004 [2]), but not everyone is suitable for cartilage. Some of them neither homogenize the cartilage, while others are producing a lot of foam during disruption process. After trying some of the currently available methods, we chose the MagNA Lyser Instrument from Roche to disrupt the cartilage and further isolate RNA by using the Fibrous Tissue Kit from Qiagen. After RNA isolation, cDNA synthesis was performed by additionally adding RNA from bacteriophage MS2 for stabilization purposes. For the RTqPCR bovine primers were designed and tested for efficiency to confirm that the whole gene expression analysis is working. Our protocol explains a whole method to perform gene expression analysis from bovine cartilage, but can also be used for human or any other animal tissue.

Hard ticks are important vectors of DNA- and RNA-based infectious microorganisms, but they also host complex microbial communities in which pathogens and symbionts can interact among each other and with the arthropod host itself. Molecular investigations on ticks and their hosted microorganisms are important for human and animal health. These analyses often imply the use of both DNA and RNA, with prompt preservation of nucleic acids after collection, and safe handling in case of low-level containment. Several commercial kits are available for the co-extraction of DNA and RNA; however, cost can be a limiting factor for the choice of this method, which also require additional reagents for nucleic acids preservation before extraction. Additionally, extraction buffers provided in commercial kits do not guarantee the safe handling in case of hazardous biological material. With the aim of epidemiological screenings for tick-borne pathogens and gene expression analyses focused on the relationship among ticks and their microbial communities, an optimized protocol for DNA and RNA co-extraction from single adult hard tick specimens (Ixodidae) has been developed using TRIzolⓇ LS Reagent.A method for•DNA/RNA co-extraction from adult hard tick specimens;•Safe sample handling;•Obtaining DNA and RNA simultaneously for diagnostic procedures and RNA for gene expression/transcriptomic analyses.

Zebrafish is a powerful animal model used to study vertebrate embryogenesis, organ development and diseases (Gut et al., 2017) [1]. The usefulness of the model was established as a result of various large forward genetic screens identifying mutants in almost every organ or cell type (Driever et al., 1996; Haffter et al., 1996) [[2], [3]]. More recently, the advent of genome editing methodologies, including TALENs (Sander et al., 2011) [4] and the CRISPR/Cas9 technology (Hwang et al., 2013) [5], led to an increase in the production of zebrafish mutants. A number of these mutations are homozygous lethal at the embryonic or larval stages preventing the generation of homozygous mutant zebrafish lines. Here, we present a method allowing both genotyping and phenotype analyses of mutant zebrafish larvae from heterozygous zebrafish incrosses. The procedure is based on the genotyping of the larval tail after transection, whereas phenotypic studies are performed on the anterior part of the zebrafish larvae. •The method includes (i) a protocol for genotyping, (ii) protocols for paraffin embedding and histological analyses, (iii) protocols for protein and histone extraction and characterization by Western blot, (iv) protocols for RNA extraction and characterization by RT-PCR, and (v) protocols to study caudal spinal cord regeneration.•The technique is optimized in order to be applied on single zebrafish embryos and larvae.

High-quality RNA is required for accurate gene expression and transcriptome analysis. The current methods of RNA extraction from berry fruits are either time-consuming or expensive. To simplify the conventional phenol-chloroform based RNA extraction method, we modified the protocol with less steps as well as the removal of the use of phenol. In this protocol, the extraction buffer is composed of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and Dithiothreitol (DTT). The method facilitates efficient removal of polysaccharides and phenolic compounds from both fruit pulp and fruit peel. Additionally, the protocol is phenol-free and less toxic than traditional phenol-containing method. High-quality RNA, with RNA Integrity Number value > 8, isolated by this method is applicable for RNA sequencing and qPCR. Only 3–4 working hours are required for one batch of RNA isolation. • Our method replaces the use of phenol-chloroform with chloroform, making the extraction less toxic. • The bilberry friut RNA is of high-quality and purity with less time input.