Precipitation Of RNA Research Articles

Selective precipitation of RNA with linear polyacrylamide

Selective precipitation of RNA is often used in molecular biology as one of the methods for separation of nucleic acids to obtain samples enriched with DNA or RNA molecules alone or for purification of RNA samples. In the present study a simple and fast approach for selective precipitation of RNA with linear polyacrylamide is proposed for the first time. The method is based on the different predispositions of the DNA and RNA molecules to bind with the polyacrylamide. In this process, the linear polyacrylamide is used as the flocculant, collecting RNA particles to form aggregate, which then precipitated at low alcohol concentration. During and after precipitation the temperature is adjusted to maintain high solubility of DNA and other contaminates at given pH, salt and alcohol concentrations on the one hand, and globular state of polyacrylamide, preventing solubility of the RNA-LPA aggregate, on the other hand. The precipitated RNA can be used directly for RT-qPCR assay. The principal advantage of the present approach is the fast and quantitative precipitation of most RNA species from very dilute solutions. This makes it possible to obtain both almost DNA-free RNA and RNA-free DNA samples in one process. Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.2007397 .

Evaluation of the RNA extraction methods in different Ginkgo biloba L. tissues

Understanding the essential processes of heredity, mechanisms of metabolism, and evolutionary traits of this ginkgo (Ginkgo biloba L.) in terms of molecular biology is necessary. The extraction of high-quality and high-integrity RNA from ginkgo is one of the basic techniques of molecular biology. Therefore, this study aimed to compare the efficacy of the six commonly used RNA extraction methods for detection. TRIzol, modified TRIzol, cetyltrimethylammonium bromide (CTAB), modified CTAB, sodium dodecyl sulfate (SDS), and RNeasy Plant Mini Kit (Qiagen) methods, were evaluated for their capacity to extract high-quality and high-quantity RNA from the embryo and endosperm of ginkgo. The advantages and disadvantages of each method were analyzed to evaluate the isolation process. The optimized modified CTAB method for embryo prevented the precipitation of RNA for a long period and reduced complex separation and precipitation processes. In addition, the modified TRIzol method is more effective in endosperm. Obvious differences in RNA quality and quantity were observed between the embryo and endosperm when the same method is used. Modified TRIzol, modified CTAB, SDS and RNeasy Plant Mini Kit (Qiagen) methods were applied to RNA extraction from other different ginkgo tissues. The modified CTAB is efficient and recommended for the isolation of good-quality total RNA from all tissues with reduced chemical use, costs, and RNA loss. The results of quantitative real-time PCR confirmed that modified CTAB methods could obtain showing low Ct values and high amplification efficiency and enabled the successful isolation of RNA from all tissues.

A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein To Facilitate the Detection of SARS-CoV-2.

Management of the coronavirus disease 2019 (COVID-19) pandemic requires widespread testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among them RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here, we propose an alternative method we call PEARL (precipitation-enhanced analyte retrieval) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers performance comparable to that of commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

Precipitation of RNA with Ethanol.

Purified RNA may need to be concentrated by precipitation for downstream applications. Precipitation of RNA with ethanol (or isopropanol) is the standard method to recover RNA from aqueous solutions.

High-quality RNA isolation from pigment-rich Dendrobium flowers.

Isolation of high-quality RNA from Dendrobium flowers is challenging because of the high levels of pigment, polysaccharides, and polyphenols. In the present study, an efficient CTAB method for RNA extraction from the pigment-rich flowers of Dendrobium was optimised. The optimised method yielded high quantities of RNA (10.1-12.9µg/g). Spectrophotometric values of A260/280 in the range of 2.2 to 2.4 and A260/230 values of 2.0 suggested that the isolated RNA was free of polyphenols, polysaccharides, and protein contaminants. RNA integrity numbers determined by microfluidics were in the range of 7.9-8.9 indicative of intact RNA. In the improved method, the addition of 3M NaCl and 3% PVP-10 in the extraction buffer, followed by an incubation period of 45min at 65°C, eliminated most of the polysaccharides, polyphenolic compounds, and denatured protein. Extraction with phenol:chloroform:isoamyl alcohol (125:24:1) effectively removed pigments from the aqueous phase, while the precipitation of RNA with lithium chloride minimised the co-precipitation of protein, DNA, and polysaccharide and resulted in the extraction of high quality of RNA. The suitability of the RNA for downstream processing was confirmed via RT-PCR amplification of Chalcone synthase gene from cDNA prepared from RNA isolated from different developmental stages of the flower of a Dendrobium hybrid. The present method will be highly useful for the isolation of RNA from pigment, polyphenol, and polysaccharide-rich plant tissues.

Inexpensive metagenomic DNA extraction protocol with high quality from marine sediments contaminated by petroleum hydrocarbons.

Marine environments are a reservoir of relevant information on dangerous contaminants such as hydrocarbons, as well as microbial communities with probable degradation skills. However, to access microbial diversity, it is necessary to obtain high-quality DNA. An inexpensive, reliable, and effective metagenomic DNA (mgDNA) extraction protocol from marine sediments contaminated with petroleum hydrocarbons was established in this study from modifications to Zhou's protocol. The optimization included pretreatment of sediment with saline solutions for the removal of contaminants, a second precipitation and enzymatic degradation of RNA, followed by purification of mgDNA extracted by electroelution. The results obtained indicated that the modifications applied to 12 sediments with total petroleum hydrocarbon (TPH) concentrations from 22.6-174.3 (µg/g dry sediment) yielded 20.3-321.3 ng/µL mgDNA with A260/A280 and A260/A230 ratios of 1.75 ± 0.08 and 1.19 ± 0.22, respectively. The 16S rRNA amplification confirmed the purity of the mgDNA. The suitability of this mgDNA extraction protocol lies in the fact that all chemical solutions utilized are common in all molecular biology laboratories, and the use of dialysis membrane does not require any sophisticated or expensive equipment, only an electrophoretic chamber.

RNA immunoprecipitation technique for Drosophila melanogaster S2 cells

RNA-binding proteins play an important role in RNA metabolism, especially in mRNA biogenesis and subsequent expression patterns regulation. RNA immunoprecipitation (RIP) is a powerful tool for detecting protein-RNA associations. In this paper, we briefly cover the history of this method for analyzing RNA-protein interactions and reviewing a number of modifications of the RIP technique. We also present an adjusted RIP protocol that was modified for Drosophila S2 cell culture. The use of this protocol allows one to perform the efficient precipitation of RNA-protein complexes and harvest RNA in amounts that are sufficient for its downstream analysis.

Improved isolation of good-quality total RNA from the optic stalk of Mud crab, Scylla paramamosain

An improved and efficient protocol was developed based on the TaKaRa RNAiso Plus Kit (Code: D9108A) for isolating good-quality total RNA from the optic stalk of mud crab, Scylla paramamosain . The protocol was based on the Trizol method with modifications. The carapace overlapping the optic stalk was retained with RNA in regular protocol. In order to remove the abundant deposition correlative with the carapace which makes the isolation of RNA particularly difficult, 5M potassium acetate solution (pH = 6.0) was added before the precipitation of RNA, and the temperature of RNA deposition was also decreased to -70oC to ensure the stabilization of RNA. Good-quality total RNA from the optic stalk of S. paramamosain could be easily isolated with this modified protocol and three conventional methods were also employed to confirm the quality of RNA. This improved method would be helpful in facilitating molecular research of crabs involving RNA from the optic stalk.

Ethanol Precipitation of RNA and the Use of Carriers

Recovery of deproteinized RNA almost always is performed via precipitation with ethanol. If the RNA is free of contaminants (i.e., it is well extracted) as determined by spectrophotometry, it will resuspend easily and be ready for any subsequent manipulation. Ethanol precipitation is quite useful because it provides quantitative recovery of any-sized RNA, from several kilobases to 20 nucleotides (nt). Major considerations in this protocol are RNA concentration, pellet identification, and resuspension.

Angiogenin-induced tRNA-derived Stress-induced RNAs Promote Stress-induced Stress Granule Assembly

Angiogenin (ANG) is a secreted ribonuclease that cleaves tRNA to initiate a stress-response program in mammalian cells. Here we show that ANG inhibits protein synthesis and promotes arsenite- and pateamine A-induced assembly of stress granules (SGs). These effects are abrogated in cells transfected with the ANG inhibitor RNH1. Transfection of natural or synthetic 5'- but not 3'-tRNA fragments (tRNA-derived stress-induced RNAs; tiRNAs) induces the phospho-eukaryotic translation initiation factor 2alpha-independent assembly of SGs. Natural 5'-tiRNAs but not 3'-tiRNAs are capped with a 5'-monophosphate that is required for optimal SG assembly. These findings reveal that SG assembly is a component of the ANG- and tiRNA-induced stress response program.

Isolation of good quality RNA from a medicinal plant seabuckthorn, rich in secondary metabolites

Medicinal plants are being widely investigated owing to their ability to produce molecules of therapeutic significance. Isolation of good quality RNA is a tedious but primary step towards undertaking molecular biology experiments. However, medicinal plants are rich in secondary metabolites and not amenable to standard RNA isolation protocols involving Guanidine isothiocyanate (GITC). So an RNA isolation protocol from difficult samples (richer in secondary metabolites) is of highest desiderata. Here we propose a new protocol suitable for isolating RNA from plant tissues rich in secondary metabolites. To standard CTAB (Cetyl Trimethyl Ammonium Bromide) buffer, addition of 2% PVPP (polyvinyl polypyrrolidone) and 350 mM beta-mercaptoethanol was found useful. Use of glacial acetic acid (1M) along with ethanol for precipitation after phenolization and chloroform extraction enhanced the RNA yield. This is the first report of using glacial acetic acid in a CTAB based protocol for the precipitation of RNA. This protocol has been validated in medicinal plant Hippophae rhamnoides vern. seabuckthorn, where standard RNA isolation methods involving GITC and TRIZol extraction buffers failed. The RNA isolated by this method was of good quality as gauged by spectrophotometric readings and denaturing agarose gel electrophoresis. To the best of our knowledge, this RNA isolation protocol has never been published before. The RNA thus obtained could be suitably used for the downstream molecular procedures like Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real Time-PCR, cDNA library construction, etc.

Rapid and Effective Method of RNA Isolation from Green Microalga Ankistrodesmus convolutus

The rapid and effective method for the isolation of RNA from green microalga Ankistrodesmus convolutus based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride is developed. This procedure avoids the use of toxic chaotropic agents and phenol while high concentration of dithiothreitol is used to inhibit RNase activity and prevent oxidative cross-linking of nucleic acids by phenolics. The extraction procedure was able to produce high quality and intact RNA from A. convolutus. The yield of total RNA was 0.69-0.73 mg/g of fresh weight, with A(260)/A(280) ratio of 1.79-1.86. The obtained RNA was of sufficient quality and suitable for downstream application such as RT-PCR and cDNA library construction. The procedure may also have wider applicability for total RNA isolation from other green microalgae species.

An optimised, small‐scale preparation of high‐quality RNA from dry seeds of Davidia involucrata

The dormancy of Davidia involucrata seeds normally lasts for an extended period of time and because of this unique property the species is an excellent model for studying the molecular mechanisms of plant dormancy. The use of minimal plant material is desirable for RNA extraction since D. involucrata is a rare plant and it is relatively difficult to collect large amounts of seeds in order to perform molecular biology studies. To improve the quality of RNA obtained from seeds of D. involucrata by eliminating the oxidation of polyphenols during extraction and by preventing polysaccharides and other impurities from being extracted. A previously described method was modified by the addition of 4% (w/v) poly(N-vinyl-2-pyrrolidone) to the dry seeds during grinding and by adding 5% (v/v) beta-mercaptoethanol and 28% (v/v) ethanol to the extraction buffer. Two further centrifugation steps (5000 and 8000 rpm) were utilised and one-seventh volume of ethanol was incubated with the supernatant at 4 degrees C for 2-3 h prior to the precipitation of RNA. Following these modifications, an effective method was established for total RNA extraction from a small amount of dry seeds of D. involucrata. The isolated RNA was shown to have high purity and integrity by gel electrophoresis and spectrophotometry, and was confirmed to be suitable for RT-PCR and the construction of cDNA libraries. The modified method reduced the amount of seeds required for extraction of total DNA and was beneficial for preserving the endangered species.

Sequential CaCl 2, polyethylene glycol precipitation for RNase-free plasmid DNA isolation

Functional genomics is facilitated by the ability to express genes in heterologous systems. In some cases function can be assayed by generation of in vitro transcripts of the unknown genes and expressing those transcripts in various expression systems. Plasmids bearing phage promoters are used to generate in vitro transcripts. Therefore, it is important to ensure that the template plasmid DNA is not contaminated with RNase from the isolation procedure. We have developed a plasmid purification protocol that does not utilize RNase yet yields pure plasmid DNA. The protocol combines the selective precipitation of RNA with 1.4 M CaCl 2, followed by a final selective precipitation of the plasmid DNA in a 10% polyethylene glycol (PEG), 250 mM NaCl solution. Purity of the resulting plasmid DNA was determined spectrophotometrically and by gel electrophoresis. No detectable contaminating RNA was observed in the plasmid DNA preparations. Inhibitory effects of the protocol were assayed by performing restriction analyses, sequencing, PCR, and in vitro transcription. These procedures were successful. The in vitro transcripts visualized by gel electrophoresis were found to be full length, thus indicating no significant endogenous RNase activity associated with the procedure.

Differential interactions of plasmid DNA, RNA and endotoxin with immobilised and free metal ions

Separation of negatively charged molecules, such as plasmid DNA (pDNA), RNA and endotoxin forms a bottleneck for the development of pDNA vaccine production process. The use of affinity interactions of transition metal ions with these molecules may provide an ideal separation methodology. In this study, the binding behaviour of pDNA, RNA and endotoxin to transition metal ions, either in immobilised or free form, was investigated. Transition metal ions: Cu 2+, Ni 2+, Zn 2+, Co 2+ and Fe 3+, typically employed in the immobilised metal affinity chromatography (IMAC), showed very different binding behaviour depending on the type of metal ions and their existing state, i.e. immobilised or free. In the alkaline cell lysate, pDNA showed no binding to any of the IMAC chemistries tested whereas RNA interacted significantly with Cu 2+-iminodiacetic acid (IDA) and Ni 2+-IDA but showed no substantial binding to the rest of the IMAC chemistries. pDNA and RNA, however, interacted to varying degrees with free metal ions in the solution. The greatest selectivity in terms of pDNA and RNA separation was achieved with Zn 2+ which enabled almost full precipitation of RNA while keeping pDNA soluble. For both immobilised and free metal ions, ionic strength of solution affected the metal ion-nucleic acid interaction significantly. Endotoxin, being more flexible, was able to interact better with the immobilised metal ions than the nucleic acids and showed binding to all the IMAC chemistries. The specific interactions of immobilised and/or free metal ions with pDNA, RNA and endotoxin showed a good potential, by selectively removing RNA and endotoxin at high efficiency, to develop a simplified pDNA purification process with improved process economics.

Extraction of totle RNA from dry Grimmia pilifera

An improved SDS/acid and phenol method was presented here to be suitable for isolation of totle RNA from dry Grimmia pilifera,a plant contained abundant secondary metabolites.Using chemical SDS as detergent in the extraction buffer,LiCl precipitation of RNA was performed finally after removing of proteins,hydroxybenzene et al.by subsequent H_2O—saturated phenol,chloroform and eyewinker.The method yielded total RNA not with integrality and high purity but with simple manipulation and cheapness.It may be applied to other dry plants full of secondary metabolites.

Selective Precipitation of RNA, Supercoiled Plasmid DNA, and Open‐Circular Plasmid DNA with Different Cationic Surfactants

Precipitation of RNA and plasmid DNA in clarified lysate produced from a bacterial culture was studied using three cationic surfactants with a different number of alkyl groups in the hydrophobic tail and cationic head. All the precipitation mixtures contained 0.5 M of NaCl. The results obtained indicated that selective precipitation was achieved when different types of cationic surfactants were used. In this study, trimethyltetradecylammonium bromide (TTAB) appeared to be a promising precipitant for precipitation of supercoiled plasmid DNA.

Simple and rapid RNA extraction from freeze-dried tissue of brown algae and seagrasses

An inexpensive and rapid RNA extraction protocol for brown algae and seagrasses is presented, based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride. The protocol avoids the use of toxic chaotropic agents and phenol; high concentrations of dithiothreitol are used to inhibit RNase activity and to prevent oxidative cross-linking of nucleic acids by phenolics. A relatively high throughput of about 100 samples in 24 h can be achieved for, for example, Northern analysis. Yields of 50–200 µg g−1 fresh weight are comparable with those obtained for higher plants using commercially available kits. Tests of the extraction procedure demonstrate that high quality, intact RNA can be obtained from a variety of lyophilized brown algal tissues, even after prolonged storage at room temperature. Lyophilization is therefore suggested as an alternative to freezing tissue at −70°C to −80°C. The RNA obtained was used directly in several downstream applications to detect Fucus plastid-encoded transcripts by RNA-labelling, RT-PCR and Northern analysis.

Effect of Inhibition of the bc1 Complex on Gene Expression Profile in Yeast

Because the respiratory chain is the major site of oxidation of the reduced equivalents and of energy production in aerobic cells, its inhibition has severe impact on the cells. Communication pathways from the respiratory chain are required to allow the cell to sense the defect and respond to it. In this work, we studied changes in gene expression induced by the treatment of yeast cells with myxothiazol, an inhibitor of the bc(1) complex, an enzyme of the respiratory chain. The pattern and time-course expression of the genes resemble those of the environmental stress response, a common gene expression program induced by sudden changes in the environment. In addition, the changes were, for most of the genes, mediated through the transcription factors Msn2/4, which play a central role in the cellular response to these stresses. By using a mutant with a myxothiazol-resistant bc(1) complex, we showed that the changes of expression of the majority of the genes was caused by the inhibition of the bc(1) complex but that other stresses might be involved. The expression pattern of CTT1, coding for a cytoplasmic catalase, was further studied. The expression of this gene was largely dependent on Msn2/4 and the inhibition of the cytochrome bc(1). Addition of oxidants of NADH was found to decrease the expression of CTT1 induced by myxothiazol treatment, suggesting that the accumulation of NADH caused by the inhibition of the respiratory chain may be involved in the signaling pathway from the mitochondria to the transcription factor.

The RNA-binding Protein IMP-3 Is a Translational Activator of Insulin-like Growth Factor II Leader-3 mRNA during Proliferation of Human K562 Leukemia Cells

IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and beta-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or beta-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells.