RNASwift: A rapid,versatile RNA extraction method free from phenol and chloroform

RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. Existing RNA extraction methods typically use hazardous chemicals including phenol, chloroform and formamide which are often difficult to completely remove from the extracted RNA. RNASwift uses sodium chloride and sodium dodecyl sulphate to lyse the cells and isolate the RNA from the abundant cellular components in conjunction with solid phase extraction or isopropanol precipitation to rapidly purify the RNA. Moreover, the purified RNA is directly compatible with downstream analysis. Using spectrophotometry in conjunction with ion pair reverse phase chromatography to analyse the extracted RNA, we show that RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods. The RNASwift method yields approximately 25 μg of RNA from only 10⁸Escherichia coli cells. Furthermore, RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods.     

Circular RNAs are abundant,conserved, and associated with ALU repeats

Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a “backsplice”) and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.     

In vivo labeling and specific magnetic bead separation of RNA for biofilm characterization and stress-induced gene expression analysis in bacteria

The method of in vivo labeling and separation of bacterial RNA was developed as an approach to elucidating the stress response of natural bacterial populations. This technique is based on the incorporation of digoxigenin-11-uridine-5′-triphosphate (DIG-11-UTP) in the RNA of active bacteria. The digoxigenin fulfills a dual role as a label of de novo synthesized RNA and a target for magnetic bead separation from a total RNA extract.Depending on the growth conditions and the population's composition, the assembly rate of DIG-11-UTP ranged from 1.2% to 12.5% of the total RNA in gram-positive and gram-negative reference bacteria as well as in natural biofilms from drinking water, surface water, and lake sediment. Separation of DIG-RNA from total RNA extracts was performed with a biotinylated anti-digoxigenin antibody and streptavidin-functionalized magnetic particles. The average separation yield from total RNA extracts was about 95% of labeled RNA. The unspecific bindings of non-labeled nucleic acids were smaller than 0.2%, as was evaluated by spiking experiments with an unmarked DNA amplicon. Applicability of the method developed was demonstrated by rRNA-directed PCR-DGGE population analysis of natural biofilms and expression profiling of two stress-induced genes (vanA and rpoS) in reference bacteria.     

Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm

The development of fast, reliable and inexpensive phenol protocol is described for the isolation of RNA from bacterial biofilm producers. The method was tested on Staphylococcus aureus (S. aureus) and other biofilm-producing gram-negative microorganisms and provided the highest integrity of RNA recovery in comparison to other methods reported here. In parallel experiments, bacterial lysis with Qiagen, NucleoSpin RNAII, InnuREP RNA Mini, Trizol and MasterPure RNA extraction Kits using standard protocols consistently gave low RNA yields with an absence of integrity. The boiling method presented here yielded high concentration of RNA that was free from 16S and 23S rRNA, contained 5S RNA. Higher yields due to improved biofilm bacterial cell lysis were achieved with an added hot phenol incubation step without the need for a bead mill or the enzyme. This method when used in conjunction with the Qiagen RNeasy Mini kit, RNA isolation was a success with greater integrity and contained undegraded 16S and 23S rRNA and did not require further purification. Contaminating DNA was a problem with the RNA processing samples; we used quantitative real-time PCR (RT-qPCR) to measure the recovery of RNA from bacterial biofilm cells using the method described here.     

Evaluation of conventional and four real-time PCR methods for the detection of Leishmania on field-collected samples in Ethiopia

In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the ‘Mary kDNA PCR’ and a newly designed ‘LC kDNA PCR’ for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.     

NUCLEOLAR-DERIVED RIBONUCLEIC ACID IN CHROMOSOMES, NUCLEAR SAP, AND CYTOPLASM OF CHIRONOMUS TENTANS SALIVARY GLAND CELLS

The distribution of monodisperse high molecular weight RNA (38, 30, 28, 23, and 18S RNA) was studied in the salivary gland cells of Chironomus tentans. RNA labeled in vitro and in vivo with tritiated cytidine and uridine was isolated from microdissected nucleoli, chromosomes, nuclear sap, and cytoplasm and analyzed by electrophoresis on agarose-acrylamide composite gels. As shown earlier, the nucleoli contain labeled 38, 30, and 23S RNA. In the chromosomes, labeled 18S RNA was found in addition to the 30 and 23S RNA previously reported. The nuclear sap contains labeled 30 and 18S RNA, and the cytoplasm labeled 28 and 18S RNA. On the basis of the present and earlier analyses, it was concluded that the chromosomal monodisperse high molecular weight RNA fractions (a) show a genuine chromosomal localization and are not due to unspecific contamination, (b) are not artefacts caused by in vitro conditions, but are present also in vivo, and (c) are very likely related to nucleolar and cytoplasmic (pre)ribosomal RNA. The 30 and 23S RNA components are likely to be precursors to 28 and 18S ribosomal RNA. The order of appearance of the monodisperse high molecular weight RNA fractions in the nucleus is in turn and order: (a) nucleolus, (b) chromosomes, and (c) nuclear sap. Since both 23 and 18S RNA are present in the chromosomes, the conversion to 18S RNA may take place there. On the other hand, 30S RNA is only found in the nucleus while 28S RNA can only be detected in the cytoplasm, suggesting that this conversion takes place in connection with the exit of the molecule from the nucleus.     

Hantaan Virus Surveillance Targeting Small Mammals at Nightmare Range,a High Elevation Military Training Area,Gyeonggi Province,Republic of Korea

Rodent-borne disease surveillance was conducted at Nightmare Range (NM-R), near the demilitarized zone in northeast Gyeonggi Province, Republic of Korea, to identify hemorrhagic fever with renal syndrome (HFRS) risks for a mountainous high-elevation (500 m) military training site. Monthly surveys were conducted from January 2008-December 2009. A total of 1,720 small mammals were captured belonging to the Orders Rodentia [Families, Sciuridae (1 species) and Muridae (7 species)] and Soricomorpha [Family, Soricidae (1species)]. Apodemus agrarius, the primary reservoir for Hantaan virus (HTNV), accounted for 89.9% (1,546) of all small mammals captured, followed by Myodes regulus (4.0%), Crocidura lasiura (3.9%), Micromys minutus (1.4%), Mus musculus (0.3%), Microtus fortis (0.2%), Apodemus peninsulae (0.2%), Tamias sibiricus (0.1%), and Rattus norvegicus (<0.1%). Three species were antibody-positive (Ab+) for hantaviruses: A. agrarius (8.2%), M. minutus (4.2%), and C. lasiura (1.5%). HTNV specific RNA was detected in 93/127 Ab+ A. agrarius, while Imjin virus specific RNA was detected in 1/1 Ab+ C. lasiura. Overall, hantavirus Ab+ rates for A. agrarius increased with weight (age) and were significantly higher among males (10.9%) than females (5.1%) (P<0.0001). High A. agrarius gravid rates during the fall (August-September) were associated with peak numbers of HFRS cases in Korea that followed high gravid rates. From 79 RT-PCR positive A. agrarius, 12 HTNV RNA samples were sequenced and compared phylogenetically based on a 320 nt sequence from the G_C glycoprotein-encoding M segment. These results demonstrate that the HTNV isolates from NM-R are distinctly separated from HTNV isolated from the People’s Republic of China. These studies provide for improved disease risk assessments that identify military activities, rodent HTNV rates, and other factors associated with the transmission of hantaviruses during field training exercises.     

Properties of visna virus particles harvested at short time intervals: RNA content, infectivity, and ultrastructure

The major RNA component of Visna virus harvested at short intervals of time (5 min) is not the 60 to 70S RNA but a molecule of higher electrophoretic mobility. This RNA has been isolated and characterized. Its sedimentation coefficient is identical to that of 30 to 40S RNA subunits obtained by heat denaturation of the 60 to 70S RNA. In 1.8% acrylamide gels without agarose the electrophoretic mobility of 30 to 40S RNA subunits present in rapidly harvested virus is slightly lower than that of the subunits obtained by denaturation of the 60 to 70S RNA; after heat denaturation the mobilities are identical. These free RNA subunits present in early virus particles assemble into a 60 to 70S RNA complex as shown by following the RNA content of early virus incubated at 37 C for various lengths of time. The rate of this maturation process is slow. There is no difference between the infectivity of immature and mature virus particles. Both particles have a dense core when examined in sections of virus pellets.     

The polyadenylated RNA of zoospores and growth phase cells of the aquatic fungus,Blastocladiella

The stored poly(A) + RNA from zoospores of the aquatic fungus Blastocladiella emersonii represents 2.5% of the total RNA and has a model MW of 425,000 daltons and an average poly(A) isostich of 32 bases. The poly(A) + RNA also represents 2.5% of the total RNA from early growth phase cells and has a modal MW of 360,000 daltons and an average poly(A) isostich of 38 bases. The poly(A) + RNA from spores and 2-hr plants contains a structure resistant to RNases T₁, T₂, and A, which can be labeled with ³²PO₄ and which will bind to DBAE-cellulose. These characteristics strongly suggest that both the zoospore poly(A) + RNA and the 2-hr cell poly(A) + RNA are capped at the 5′ end; and, hence, it is unlikely that capping is involved in the control of protein synthesis during germination.Approximately 80% of the poly(A) + RNA of the spore is located in the membrane-enclosed ribosomal nuclear cap, and more than 90% of the poly(A) + RNA within the cap is found in the 80S monoribosome and heavier fractions.Synthesis of new poly(A) + RNA occurs very early during zoospore germination, and the labeled poly(A) + RNA rapidly enters the newly organized polysomes. The labeling data for early germination also suggest that cytoplasmic polyadenylation occurs.     

Ribonucleotide Sequence Homology Among Avian Oncornaviruses

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between ³H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-0, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of ³H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as efficiently (100%) as by unlabeled AMV RNA. Hybridization between ³H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between ³H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between ³H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNAs. Hybridization between ³H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 to 26% by RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybridization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0 are different from the sequences shared by AMV and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

Assessing the Quality of Hybridized RNA in Affymetrix GeneChips Using Linear Regression

The quality of data from microarray analysis is highly dependent on the quality of RNA. Because of the lability of RNA, steps involved in tissue sampling, RNA purification, and RNA storage are known to potentially lead to the degradation of RNAs; therefore, assessment of RNA quality and integrity is essential. Existing methods for estimating the quality of RNA hybridized to a GeneChip either suffer from subjectivity or are inefficient in performance. To overcome these drawbacks, we propose a linear regression method for assessing RNA quality for a hybridized Genechip. In particular, our approach used the probe intensities from the .cel files that the Affymetrix software associates with each microarray. The effectiveness and the improvements of the proposed method over the existing methods are illustrated by the application of the method to the previously published 19 human Affymetrix microarray data sets for which external verification of RNA quality is available.     

Effect of Growth Rate and Starvation-Survival on Cellular DNA, RNA, and Protein of a Psychrophilic Marine Bacterium

DNA, RNA, and protein concentrations from starved ANT-300 cell populations grown at different growth rates fluctuated corresponding to the three stages of starvation-survival on total and viable cell bases. During stage 1 of starvation-survival, two to three peaks in the concentration levels for all three macromolecules were characteristic. During stage 2, DNA per total cell dropped to between 4.2 and 8.3% of the original amount for all of the cell populations examined, and it stabilized throughout stage 3. The decrease in DNA per cell was also observed in electron micrographs of cellular DNA in unstarved compared with starved cells. The fluctuations of RNA and protein per total cell concentrations observed during stage 2 coincided in all cases, except for the cells from dilution rate (D) = 0.015 h⁻¹. This ANT-300 cell population showed a decrease in RNA per total cell to only 29.2% and an increase in protein to 129.7% of the original amount after 98 days of starvation. During stage 3, DNA, RNA, and protein concentrations per total cell also stabilized to continuous levels. Cells from the faster-growth-rate cell populations of D = 0.170 h⁻¹ and batch culture had elevated protein per total cell concentrations, which remained primarily residual during the starvation period. Starved cells from D = 0.015 h⁻¹ had estimated nucleoid and cell volumes of 0.018 and 0.05 μm³, respectively, yielding a nucleoid volume/cell volume ratio of 0.40. We consider these data to indicate that slow-growth-rate cells are better adapted for starvation-survival than their faster-growth-rate counterparts.     

Method Optimization for Extracting High-Quality RNA From the Human Pancreas Tissue

Nucleic acid sequencing is frequently used to determine the molecular basis of diseases. Therefore, proper storage of biological specimens is essential to inhibit nucleic acid degradation. RNA isolated from the human pancreas is generally of poor quality because of its high concentration of endogenous RNase. In this study, we optimized the method for extracting high quality RNA from paired tumor and normal pancreatic tissues obtained from eight pancreatic cancer patients post-surgery. RNA integrity number (RIN) was checked to evaluate the integrity of RNA, we tried to extract the RNA with an RIN value of 8 or higher that allows for the latest genetic analysis. The effect of several parameters, including the method used for tissue lysis, RNAlater treatment, tissue weight at storage, and the time to storage after surgical resection, on the quantity and quality of RNA extracted was examined. Data showed that the highest quantity of RNA was isolated using a combination of manual and mechanical methods of tissue lysis. Additionally, sectioning the tissues into small pieces (<100 mg) and treating them with RNAlater solution prior to storage increased RNA stability. Following these guidelines, high quality RNA was obtained from 100% (8/8) of tumor tissues and 75% (6/8) of normal tissues. High-quality RNA was still stable under repeated freezing and thawing. The application of these results during sample handling and storage in clinical settings will facilitate the genetic diagnosis of diseases and their subsequent treatment.