Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells

An extremely simple procedure for preparing cytoplasmic RNA from small numbers of cells is described. Cells are lysed with the detergent NP-40 and efficient extraction of protein from the postnuclear cytoplasmic lysate is ensured by denaturation with sodium dodecyl sulfate and urea. This procedure is suitable for preparing RNA from many cell types. All procedures have been scaled down to be performed in 1.5-ml microfuge tubes and thus RNA may be prepared from small numbers of cells. The procedure is extremely rapid and RNA is ready for Northern gel analysis in less than 30 min. Because so few steps are involved, RNA recovery is quantitative.     

A general purpose method for extracting RNA from Dictyostelium cells

Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods; however, it has been adjusted because of the large quantities of carbohydrate and nucleases found in Dictyostelium cells. After cell lysis and phenol:chloroform extraction, the resulting high-quality RNA isolated with the described protocol allows the molecular genetic analysis of wild-type and genetically modified cells. The purified RNA can be used for analyses such as northern blotting, RT-PCR and microarrays. This procedure requires approximately 2 h to complete.     

Quantitative detection of messenger RNA by solution hybridization and enzyme immunoassay

A novel nucleic acid detection technique is described for the quantitative measurement of eukaryotic mRNA in biological samples. The procedure involves two steps: a hybridization reaction in solution with a biotinylated cDNA probe, and a conventional enzyme immunoassay that uses a monoclonal antibody for DNA.RNA hybrids to detect the specific mRNA.cDNA complexes. The method has comparable sensitivity to 32P-based methods and yields results that are quantitative and highly reproducible. Furthermore, the test can be performed using unfractionated cytoplasm without the need for extraction with organic solvents. This technique provides a rapid and quantitative method for studying changes in cellular mRNA levels, and it is suitable for testing large numbers of samples.     

Rapid extraction of high molecular weight RNA from cultured cells and granulocytes for Northern analysis.

The study of messenger RNA in mammalian cells by Northern analysis requires the extraction of intact RNA in pure form. Although a number of reliable techniques have been developed for the purpose, most are fairly complex, involving steps such as ultracentrifugation and multiple extractions with large volumes of phenol and chloroform. When the number of cell samples to be analyzed is large, these techniques can be unwieldy. I now describe an RNA purification procedure which is simple enough to allow handling of a large number of cultured cell samples. It uses safe and inexpensive reagents and produces a high yield of pure total cell RNA, essentially free of DNA and ribonuclease, suitable for Northern analysis. The procedure also allows extraction of intact RNA from human granulocytes, cells which are rich in ribonuclease and contain very low amounts of RNA.     

A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis

Extraction of intact RNA is essential for quantitative gene expression analysis. Isolating high quality RNA from gram-positive bacteria is known to be problematic particularly from organisms that have optimal growth temperatures greater than 45 °C. We report a novel extraction protocol for the rapid isolation of fully intact RNA from thermophilic Geobacillus thermoleovorans using a lysing matrix containing a mixture of ceramic and glass beads, triisopropylnaphthalene sulfonic acid (TNS), and p-4-aminosalicyclic acid (PAS). Combining both detergents, TNS and PAS, appeared to increase denaturation of RNases at thermophilic temperatures. Gel electrophoresis revealed that only RNA isolated using the TNS-PAS procedure demonstrated sharp, undegraded 23S, 16S, and 5S ribosomal RNA bands. RNA extracted from geobacilli using commercially available kits was extensively degraded and was not suitable for detecting gene expression. Total RNA yields extracted with the TNS-PAS protocol were greater than eightfold higher than those obtained with available kits. Critically, it was also shown that only RNA isolated with the TNS-PAS-based method was suitable for monitoring thermophile gene expression patterns using RT-PCR analysis.Communicated by G. Antranikian     

Rapid methods to extract DNA and RNA from Cryptococcus neoformans

Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations.     

High-quality RNA from cells isolated by laser capture microdissection

Laser capture microdissection (LCM) provides a rapid and simple method for procuring homogeneous populations of cells. However, reproducible isolation of intact RNAfrom these cells can be problematic; the sample may deteriorate before or during sectioning, RNA may degrade during slide staining and LCM, and inadequate extraction and isolation methods may lead to poor recovery. Our report describes an optimized protocol for preparation of frozen sections for LCM using the HistoGene Frozen Section Staining Kit. This slide preparation method is combined with the PicoPure RNA Isolation Kitfor extraction and isolation of RNA from low numbers of microdissected cells. The procedure is easy to perform, rapid, and reproducible. Our results show that the RNA isolated from the LCM samples prepared according to our protocol is of high quality. The RNA maintains its integrity as shown by RT-PCR detection of genes of different abundance levels and by electrophoretic analysis of ribosomal RNA. RNA obtained by this method has also been used to synthesize probes for interrogating cDNA microarray analyses to study expression levels of thousands of genes from LCM samples.     

A METHOD FOR DETERMINING TOTAL PROTEIN OF ISOLATED CELLULAR ELEMENTS AND CORRESPONDING TRITIUM RADIOACTIVITY

A method is described for the microanalysis of protein, obtained from isolated tissue elements, in the range of 500 µµg-500 mµg. The method entails solubilization of cellular protein with phosphoric acid and heat after extraction of acid-soluble compounds, lipids, and RNA. A procedure for the extraction and recovery of cellular RNA by the use of 40% trichloroacetic acid is presented. The solubilized protein, in the form of a microdroplet, is photomicrographed with monochromatic light at 230 mµ. Total density in the microdroplet is determined from calibrated photographic plates by microdensitometry, and is converted to protein mass by using an experimentally determined average specific absorbance value. A solubilized protein labeled with tritium can be recovered after photomicrography, combusted, and reduced to generate tritiated gas for high-efficiency tritium radiometry. Total protein was analyzed in (a) nerve cells of three different sizes from Deiters' nucleus of the rabbit; and the whole rod cell and rod cell nucleus of the rabbit retina.     

从少量培养细胞中同时提取微量蛋白和RNA的方法探讨

为建立一项从少量培养细胞中同时提取RNA和蛋白质的技术 ,向 2～ 3× 10 5细胞中加入 1ml自制RNA提取试剂 ,RNA抽提后剩下的中下两相 ,用异丙醇、盐酸胍和无水乙醇抽提蛋白质 .同时用进口Tripure试剂、经典的异硫氰酸胍 苯酚 氯仿一步抽提RNA法和分子克隆实验手册裂解液制备蛋白质的方法 ,作为对照 .自制试剂提取的总RNA ,18S、2 8S清晰可见 ,2 8S比 18S带亮度强 2～ 3倍 ,带与带之间无拖尾现象 ,5S隐约可见 ,而且成功地进行了Northern印迹、RT PCR分析 ,与经典方法差异不大 ;用此法所提蛋白质 ,经SDS PAGE检测 ,蛋白分离效果很好 ,无杂质 ,且Western印迹检测Giα蛋白 ,可见一条清晰的特异带 ,与常规提取蛋白质 ,结果相似 .从微量细胞中同时提取的RNA和蛋白质 ,得率高、纯度好 ,具有化学完整性和生物学性质     

Rapid isolation of RNA using proteinase K and sodium perchlorate.

A simple, efficient procedure for the isolation of cellular nucleic acids is described. It combines the use of sodium dodecyl sulfate, proteinase K, sodium perchlorate, and isopropanol precipitation. The yields and purity of RNA extracted from a variety of sources are comparable or superior to those obtained by phenol extraction. High molecular weight RNA (ribosomal as well as nonribosomal) is recovered intact and in high yield. Fibroin messenger RNA (M_r 5.8 × 10⁶) isolated by this procedure is biologically active.     

DNA Extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction

This article describes two procedures for the purification of genomic DNA from small blood volumes of whole blood using DNAzol^®BD. In the first procedure, DNA is isolated from 1–20 μL of whole blood using a fast and simple protocol that is appropriate for the simultaneous extraction of a large number of samples. The isolated DNA is suitable for gel electrophoresis and polymerase chain reaction (PCR). In the second procedure, cellulose blood cards containing approx 5 μL of dried blood are treated with DNAzol BD in order to retain DNA on the cellulose matrix while removing other cellular components. The blood card with DNA subsequently serves as template in PCR. The blood card processing and amplification procedures are performed in the same PCR tube without any centrifugation steps, making the combined procedures amenable for automated DNA preparation and amplification in a single tube.     

Simultaneous purification of DNA and RNA from small numbers of eukaryotic cells

An extraction procedure for the simultaneous isolation of RNA and DNA from tissue culture cells is described. The procedure is a variation of the guanidium/lithium chloride method for RNA isolation which is rapid, simple, and avoids costly ultracentrifugation equipment. The genomic DNA yielded by this procedure is greater than 50 kb in length and may be readily cleaved by restriction endonucleases. Sufficient DNA for Southern blot analysis, and RNA for Northern blot or nuclease protection analysis, can be obtained from as few as 2 x 10(6) cells, making this method particularly suitable for the genetic screening of large numbers of individual, stably transfected cell clones.     

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure

A new one-step procedure for the isolation of bacterial RNA, involving lysis by proteinase K in the presence of sodium dodecyl sulfate, is described. Pulse-labeled RNA isolated by this procedure for Bacillus brevis, Bacillus subtilis, and Escherichia coli B has been found to contain a substantial fraction (15-40%) of polyadenylated RNA as determined by adsorption to oligo(dT)-cellulose. This contrasts with RNA isolated by procedures involving phenol extraction, a process which appears to lead to the selective loss of polyadenylated RNA. The presence of polyadenylated RNA in E. coli was confirmed by an independent method which involved hybridization with [3H]polyuridylic acid. Using the proteinase K method for RNA isolation, it was possible to demonstrate the in vitro synthesis of polyadenylated RNA by toluene-treated cells of B. brevis, B. subtilis, and E. coli.     

Does extraction of DNA and RNA by magnetic fishing work for diverse plant species?

An automated nucleic acid extraction procedure with magnetic particles originally designed for isolation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from animal tissues was tested for plant material. We isolated genomic DNA and total RNA from taxonomically diverse plant species representing conifers (Scots pine), broad-leaved trees (silver birch and hybrid aspen), dwarf shrubs (bilberry), and both monocotyledonous (regal lily) and dicotyledonous (Saint John's wort, round-leaved sundew, and tobacco) herbaceous plants. Buffers developed for DNA extraction were successfully used in addition to manufacturer's extraction kits. The quality of RNA was appropriate for many applications, but the quality of DNA was not always sufficient for polymerase chain reaction (PCR) amplification. However, we could strikingly improve the quality by eliminating the adherent compounds during the extraction or later in the PCR phase. Our results show that the use of the procedure could be extended to diverse plant species. This procedure is especially suitable for small sample sizes and for simultaneous processing of many samples enabling large-scale plant applications in population genetics, or in the screening of putative transgenic plants.     

Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique.

A rapid method of determining simultaneously DNA and RNA in mammary gland homogenates using the ethidium bromide technique is discussed. The method utilizes a quantitative extraction of DNA and RNA with 2.0m sodium chloride, SDS, and EDTA at pH 8.0. Assays of mammary gland RNA and DNA using previously published methods were compared with determinations using the ethidium bromide technique. While the fluorescence method gave lower values for RNA when compared to those obtained using the orcinol or absorbancy ratio (OD 260nm/280nm), DNA measurements agreed well with the values determined by the diphenylamine technique. Extinction coefficient data for total mammary gland RNA isolated using a modified phenol extraction procedure are also presented.     

A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds

Extraction of high-quality RNA from Arabidopsis seeds has been a challenge. Here we report a two-step TRIzol-based procedure for RNA extraction from Arabidopsis siliques and dry seeds. This procedure employs a modified, high pH (pH 9.5) extraction buffer. High pH plus the addition of either DTT or β-mercaptoethanol in the extraction buffer effectively inhibits RNase activity during the extraction, and removes most polysaccharides, polyphenols and other insoluble material. TRIzol reagent was subsequently used to purify the RNA. Using this procedure we isolated high-quality DNA-free RNA samples without DNase I treatment from Arabidopsis seeds or siliques in less than 3 h.     

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA,a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

AIMS: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. METHODS AND RESULTS: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. CONCLUSIONS: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.     

Cell-free translation of messenger RNAs from adult and fetal human muscle. Characterization of neosynthesized glycogen phosphorylase, phosphofructokinase and glucose phosphate isomerase

Using a procedure of ethanol precipitation in concentrated guanidine . HCl solutions followed by chloroform/isoamylic alcohol extraction and washing in 3 M sodium acetate, we isolated high-molecular-weight cellular RNA from human fetal and adult skeletal muscle. About 500 micrograms RNA were obtained/g of fetal muscle and 50 micrograms RNA/g of adult muscle. Both RNA preparations were efficiently translated in a cell-free reticulocyte lysate system and directed synthesis of various polypeptides, one of them of Mr 200,000 probably corresponding to myosin heavy chains. Dodecylsulphate/polyacrylamide gel electrophoretic pattern of polypeptides neosynthesized using either fetal or adult RNA exhibited several differences. Three neosynthesized cytosolic muscle enzymes were purified from the translation mixtures using a micro-method of immunoaffinity chromatography; specificity of the neosynthesized polypeptides purified according to this procedure was checked by immunological competition with the corresponding unlabeled pure muscle enzymes. Successful cell-free translation of RNA from adult skeletal muscle and purification of neosynthesized human enzymes are reported for the first time. These methods, indispensable for further studies on human adult muscle gene expression, could also shed light on the mechanism of some inherited molecular diseases and tumoral or dystrophic processes.