Rapid procedure for the isolation of high molecular weight RNA and DNA from yeast using lyticase and sodium dodecyl sulfate

Lyticase, an enzyme preparation isolated from the culture supernatant of Oerskovia xanthineolytica, was found to lyse yeast cells in the presence of sodium dodecyl sulfate (SDS). Undegraded nucleic acids could be isolated from the lysate demonstrating the usefulness of the described procedure for the rapid isolation of high molecular weight RNA and DNA from whole cells.     

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.     

Transfection of RNA from Organ Samples of Infected Animals Represents a Highly Sensitive Method for Virus Detection and Recovery of Classical Swine Fever Virus

Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.     

Preservation of algal and higher plant ribosomal RNA integrity during extraction and electrophoretic quantitation

Isolation of high molecular weight ribosomal RNA from the wall-less alga Olisthodiscus luteus and the angiospermous plant Sauromatum guttatum is described. It has been found that a buffer which contains magnesium must be used to successfully isolate Olisthodiscus rRNA whereas the isolation of intact Sauromatum rRNA requires a buffer system containing a high amount of the chelator EDTA, Sauromatum but not Olisthodiseus extracts were contaminated with ribonuclease unless the inhibitor diethylpyrocarbonate was used during the ribonucleic acid extraction procedure. Nuclease levels were monitored by coincubating [³H]-labeled Escherichia coli ribosomal RNA with the experimental RNA samples. The effects of detergents on the isolation and quantitation of RNA are presented, and methods to avoid loss of highly thermolabile plant ribosomal RNA species are discussed.     

Isolation of High Quality RNA from Oilseeds of Jatropha curcas

High quality RNA with good yield is a prerequisite for carrying out several molecular biology studies. Recalcitrant tissues such as oilseeds pose several problems while isolating good quality RNA. We have standardized a fast and simple protocol for RNA isolation from the seeds of Jatropha curcas, which gives good quality RNA without compromising for the yield. By including pre wash of seed powder with acetone and removal of polysaccharides through selective precipitation, we have been regularly isolating good quality total RNA in the range of 300–450 μg g^?1 depending upon tissue type. The RNA isolated by this procedure is devoid of any contaminating DNA. The RNA preparations have been subjected to cDNA synthesis and PCR, and found suitable for these studies. This method also works satisfactorily with groundnut and mustard seeds.     

Isolation of High-Quality RNA from <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>, a Desert Plant Rich in Secondary Metabolites

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.     

Rapid immunomagnetic negative enrichment of neutrophil granulocytes from murine bone marrow for functional studies in vitro and in vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*10⁶ PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.     

Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet-containing cells, which is useful for isolation of the androgen-producing Leydig cells. Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis.     

Exportin-5 orthologues are functionally divergent among species

Exportin-5, an evolutionarily conserved nuclear export factor belonging to the importin-β family of proteins, is known to play a role in the nuclear export of small noncoding RNAs such as precursors of microRNA, viral minihelix RNA and a subset of tRNAs in mammalian cells. In this study, we show that the exportin-5 orthologues from different species such as human, fruit fly and yeast exhibit diverged functions. We found that Msn5p, a yeast exportin-5 orthologue, binds double-stranded RNAs and that it prefers a shorter 22 nt, double-stranded RNA to ~80 nt pre-miRNA, even though both of these RNAs share a similar terminal structure. Furthermore, we found that Drosophila exportin-5 binds pre-miRNAs and that amongst the exportin-5 orthologues tested, it shows the highest affinity for tRNAs. The knockdown of Drosophila exportin-5 in cultured cells decreased the amounts of tRNA as well as miRNA, whereas the knock down of human exportin-5 in cultured cells affected only miRNA but not tRNA levels. These results indicate that double-stranded RNA binding ability is an inherited functional characteristic of the exportin-5 orthologues and that Drosophila exportin-5 functions as an exporter of tRNAs as well as pre-miRNAs in the fruit fly that lacks the orthologous gene for exportin-t.     

An alternative Candida spp. cell wall disruption method using a basic sorbitol lysis buffer and glass beads

This report describes a modified, cost-effective method of cell wall disruption for the yeast Candida spp., which employs the use of glass beads in a simple sorbitol lysis buffer. This method can be used in conjunction with a commercial RNA or genomic DNA isolation method to obtain high-quality RNA or DNA.     

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A₂₆₀/A₂₈₀ ratios ranged from 1.90 to 2.04 and A₂₆₀/A₂₃₀ ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.