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At least 379 records · Page 21Linked to original sources
PubMed2002

Efficient isolation of total RNA from antibiotic-producing bacterium Amycolatopsis mediterranei.

RNA extraction from antibiotic-producing actinomycetes can be a difficult and time-consuming process due to their special peptidoglycans cell wall composition and the short life of RNA. Hence, the rapidity of cellular lysis and complete inhibition of RNase are of particular importance for isolating intact RNA of high quality. The genus of Amycolatopsis mediterranei produces many clinically important antibiotics, such as rifamycin and vancomycin; however, the available methods for bacterial RNA isolation did not work very well with this genus. In this report, we described a new method for RNA isolation using the combination of LiCl, urea and guanidinium thiocyanate to disrupt the cell wall of Amycolatopsis. Compared with earlier published RNA isolation methods, the method gave higher yields of pure and intact RNA. About 1 microg total RNA free of DNA contamination can be obtained from 1 mg wet weight of A. mediterranei. The integrity of the RNA was demonstrated by formaldehyde agarose gel electrophoresis and Northern blot analyses.

Actinomycetales↗
PubMed2002

Quantitative gene expression analysis in renal biopsies: a novel protocol for a high-throughput multicenter application.

BACKGROUND: Recent advances in gene expression analysis may add the quantification of mRNA species in renal biopsies to routine diagnostic procedures in nephrology. METHODS: A systematic evaluation was performed on the relevant steps required to efficiently obtain cDNA from renal biopsies for high-throughput reverse transcription-polymerase chain reaction (RT-PCR) based mRNA quantification. RESULTS: The protocol preserves mRNA integrity by a novel RNase inhibitor and allows meticulous microdissection followed by maximal RNA recovery from tissue samples. Reverse transcription was optimized to give the best yield from minimal starting material. RNA quantity and quality were systematically investigated by real-time RT-PCR and electrophoresis on a microfluidic system, respectively. The reported procedure offers high RNA preservation and increases the yield of cDNA significantly compared to former protocols. CONCLUSION: The simplicity of biopsy material acquisition combined with the centrally performed processing makes this protocol suitable for a wide spectrum of expression analysis in diverse clinical settings.

Biopsy↗
PubMed2005

QIAamp MinElute virus kit effectively extracts viral nucleic acids from cerebrospinal fluids and nasopharyngeal swabs.

BACKGROUND: Nucleic acid preparation from a variety of clinical specimens requires efficient target recovery and amplification inhibitor removal and is critical for successful molecular diagnosis. The QIAamp MinElute Virus kit (Qiagen Inc., Valencia, CA) was compared to the two existing methods currently used in our laboratory, IsoQuick (Orca Research Inc., Bothell, WA) for DNA extraction and RNAzol B (Leedo Laboratories Inc., Houston, TX) for RNA extraction, of viral nucleic acids. STUDY DESIGN: A total of 150 clinical specimens, including cerebrospinal fluid (CSF) and nasopharyngeal swabs (NPS), were used to determine the extraction efficiency of the MinElute compared to the other two methods. Nucleic acid recovery, hands-on time, turn-around-time and cost were compared across all kits. RESULTS: There was complete concordance between the MinElute and IsoQuick/RNAzol kits when herpes simplex virus (HSV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), influenza A virus or enteroviruses were detected using a colorimetric microtiter plate PCR system. The kits were equivalent in their ability to detect either DNA or RNA with superior ability to recover a high quality and quantity of RNA. With the potential to process larger specimen volumes, the MinElute kit can significantly shorten processing time from 2h to 50-55min. CONCLUSIONS: Although relatively high test kit costs were noted, the MinElute kit provides another rapid and user-friendly specimen processing tool in the diagnostic molecular microbiology laboratory.

Cerebrospinal Fluid↗
PubMed2006

The application of laser microdissection to in planta gene expression profiling of the maize anthracnose stalk rot fungus Colletotrichum graminicola.

Laser microdissection (LM) offers a potential means for deep sampling of a fungal plant-pathogen transcriptome during the infection process using whole-genome DNA microarrays. The use of a fluorescent protein-expressing fungus can greatly facilitate the identification of fungal structures for LM sampling. However, fixation methods that preserve both tissue histology and protein fluorescence, and that also yield RNA of suitable quality for microarray applications, have not been reported. We developed a microwave-accelerated acetone fixation, paraffin-embedding method that fulfills these requirements and used it to prepare mature maize stalk tissues infected with an Anemonia majano cyan fluorescent protein-expressing isolate of the anthracnose stalk rot fungus Colletotrichum graminicola. We successfully used LM to isolate individual maize cells associated with C. graminicola hyphae at an early stage of infection. The LM-derived RNA, after two-round linear amplification, was of sufficient quality and quantity for global expression profiling using a fungal microarray. Comparing replicated LM samples representing an early stage of stalk cell infection with samples from in vitro-germinated conidia, we identified 437 and 370 C. graminicola genes showing significant up- or downregulation, respectively. We confirmed the differential expression of several representative transcripts by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and documented extensive overlap of this dataset with a PCR-subtraction library enriched for C. graminicola transcripts in planta. Our results demonstrate that LM is feasible for in planta pathogen expression profiling and can reveal clues about fungal genes involved in pathogenesis. The method in this report may be advantageous for visualizing a variety of cellular features that depend on a high degree of histochemical preservation and RNA integrity prior to LM.

Acetone↗
PubMed1999

Validation of a rapid method for extraction of total RNA applied to Leishmania promastigotes.

This report explains a rapid procedure (approximately 50 min) for the isolation of highly purified total RNA from Leishmania promastigotes based on extraction with acidic phenol. The simplicity of the manipulations required make this method ideal for processing multiple samples; the quality of the RNA obtained is suitable for reverse transcription polymerase chain reaction analysis.

Animals↗
PubMed1995

Random primer p(dN)6-digoxigenin labeling for quantitation of mRNA by Q-RT-PCR and ELISA.

The ability to accurately measure mRNA levels in samples of total RNA is essential for studies on control of gene expression. The mRNAs from the housekeeping gene for phosphoglycerate kinase (PGK-1) can serve as a quality control for RNA samples. We describe an enzyme-linked immunosorbent assay (ELISA) method for mRNA determination by Q-RT-PCR, a quantitative reverse transcriptase-mediated PCR assay with competitive internal standards. After PCR, two biotinylated capture primers, one specific for PGK-1 cDNA and another one for internal standard, are annealed in separate assays so that each can attach DNA to a streptavidin-coated microplate. The captured DNA is either internally labeled with digoxigenin (DIG) or is "developed" after annealing with DIG-labeled primers. Bound DNA is then quantitated by adding DIG-specific antibody with attached alkaline phosphatase and measuring phosphatase activity with a chromogenic substrate and a plate reader. We compared different capturing methods and various primers labeled with DIG at their 3' ends. We determined that amplified PGK-1 DNA specifically captured with biotinylated primers was efficiently assayed with random p(dN)6-DIG.

Base Sequence↗
PubMed2003

A simple method for in vivo expression studies of Xanthomonas axonopodis pv. citri.

A major problem in studying bacterial plant pathogens is obtaining the microorganism directly from the plant tissue to perform in vivo expression (protein or mRNA) analyses. Here we report an easy and fast protocol to isolate Xanthomonas axonopodis pv. citri directly from the host plant, in sufficient amounts to perform protein fingerprinting by 2-D gel electrophoresis as well as RNA expression assays. The protein profile obtained was very similar to that of X. axonopodis pv. citri grown in the presence of a leaf extract of Citrus sinensis; however, some differential proteins expressed in vivo were observed. Total RNA extraction revealed typical 16S and 23S bands in the agarose gel, and RT-PCR reactions using primers specific for genes of the bacterium confirmed the quality of the RNA preparation. Also, RT-PCR reactions using plant ribosomal primers were employed, and no amplification product was obtained, indicating that plant RNA is not present in the bacterium RNA sample.

Bacterial Proteins↗
PubMed2000

Extraction of total RNA from a high pigment content plant. Marigold (Tagetes erecta).

The Mexican marigold (Tagetes erecta) produces inflorescences of intense yellow color that contain high levels of xanthophylls, particularly lutein, which makes it a suitable model for the study of carotenoid biosynthesis and regulation throughout the development of the inflorescences. However, these studies require the recovery of total RNA from floral buds and inflorescences at different developmental stages, each of which presents specific extraction problems. Four protocols were tested, but only through the modification of one of them was it possible to obtain total RNA of sufficient quality and quantity to perform RT-PCR and Northern blots and to construct a cDNA library. This article presents the modified protocol for the recovery of total RNA from carotenoid-rich plant tissues.

Asteraceae↗
PubMed1989

Cloning of a cDNA encoding MAP1B in rat brain: regulation of mRNA levels during development.

This article describes the isolation of a microtubule-associated protein 1B (MAP1B) cDNA clone from a rat brain lambda gt11 library and the study of MAP1B mRNA expression during brain development. On Northern blots, the cDNA hybridized with an mRNA of greater than 10 kilobases which was present only in the brain. The identity of the cDNA was confirmed by the characterization of the antiserum against the fusion protein, and also by comparing both the original antibody and the anti-fusion protein antiserum with a panel of well-studied monoclonal antibodies against different forms of MAP1 and MAP2. The regulation of MAP1B mRNA during development was studied in whole brain, cerebral cortex, hypothalamus, brainstem, and olfactory bulbs. The steady-state levels of MAP1B mRNA in all tissues examined were relatively low in the adult compared to developing brains. This decrease varied in different brain regions, and its time course appeared to coincide with the pattern of postnatal developmental and morphological events. The developmental patterns of the MAP1B mRNA and protein in the brain were similar, suggesting that expression of this protein is under transcriptional control. The RNA blots were also probed with beta-actin and beta-tubulin to compare the levels of MAP1B mRNA with other cytoskeletal elements and as controls for the quality of the RNA.

Animals↗
PubMed1997

Isolating RNA from clinical samples with Catrimox-14 and lithium chloride.

RNA is a highly informative molecule that has great potential as a target for diagnostic studies. This potential can be reached only when reliable methods for isolating RNA are available in the clinical environment. Cationic surfactants lyse cells and precipitate nucleic acids. We have described a novel cationic surfactant (tetradecyltrimethylammonium oxalate, Catrimox-14), which is particularly effective in precipitating RNA from cells and which can be applied to clinical specimens. We examine the utility of a method of recovering RNA from the surfactant-nucleic acid precipitate, in which 2 M lithium chloride is used to extract the DNA and surfactant from the precipitate; RNA (being insoluble in lithium chloride solution) remains in the pellet. The yield of RNA from peripheral blood mononuclear cells by the Catrimox-LiCl method we describe was the same yield by a conventional method using guanidine thiocyanate, phenol, and chloroform (GPC). The quality of the RNA, judged by agarose gel electrophoresis, A260/280 ratio and its ability to serve as a target for reverse transcription and PCR, was the same. RNA was isolated and amplified from blood stored for at least 2 weeks in Catrimox solution at room temperature. RNA was also easily isolated with the Catrimox-LiCl method in good yield from frozen sections of mouse liver, spleen, kidney and brain, and from core biopsies of liver and kidney. RNA isolated from needle aspirates of liver, spleen, kidney, pancreas, and brain was easily amplified by RT-PCR. The Catrimox-LiCl method is simple and does not call for the use of corrosive reagents. The Catrimox-LiCl method removes 98% of the DNA. We conclude that the Catrimox-LiCl method is suitable for use in clinical applications of RNA-based diagnosis.

Animals↗
PubMed2006

Amplification of low quantity bacterial RNA for microarray studies: time-course analysis of Leptospirillum ferrooxidans under nitrogen-fixing conditions.

We have developed a method for the amplification of low quantity total bacterial RNA for DNA microarrays analysis. Current methods are based on the linear amplification by the in vitro transcription from the T7 promoter, similar to that used for eukaryotic mRNA amplification. For the incorporation of T7 promoter, the prokaryotic RNA must be enzymatically modified for the incorporation of a polyA tail at the 3' end to emulate the eukaryotic mRNA. The method we describe and validate herein avoids this step by the direct and random incorporation of the T7 promoter. From 500 ng of total bacterial RNA, we obtained 130-150 microg of antisense RNA, such products being good substrate for fluorescent labelling and DNA microarray analysis. The method was validated with bacterial samples from which it is very difficult to obtain sufficient amounts and quality of total RNA for global gene expression analysis. This is critical for low cell density growing microorganisms, environmental samples, or many extremophiles where the composition of the cultural media severely affects the RNA yield, like in the case of the acidophile and iron oxidizer Gram-negative bacterium Leptospirillum ferrooxidans. We further validated our amplification method in parallel experiments with non-amplified RNA by following the expression of the L. ferrooxidans nif regulon along the time-course of growth.

Cluster Analysis↗
PubMed1995

A simple and efficient method for the isolation of total RNA from the fission yeast Schizosaccharomyces pombe.

A simple and efficient procedure was described for the isolation of total RNA from the fission yeast Schizosaccharomyces pombe. The present study demonstrated that the quality and the quantity of S. pombe RNA were increased by substituting phenol/chloroform mixture for phenol as a deproteinizing agent in the first vortexing step and using an ice bath instead of a dry ice-ethanol bath in the freezing step. Additionally, this protocol had the advantage of extracting total RNA without any degradation of S. pombe cells. Furthermore, the high amounts and quality of RNA extracted by this modified procedure enabled us to perform some experiments such as Northern blot, S1 mapping, primer extension, and reverse transcriptase reaction-polymerase chain reaction (RT-PCR) without further RNA purification. We suggest that this procedure is very useful to analyse primary structures and steady-state levels of RNA from S. pombe.

Base Sequence↗
PubMed2005

Laser capture microdissection of hepatic stages of the human parasite Plasmodium falciparum for molecular analysis.

Despite the sequencing of parasite genomes and development of DNA microarray technology, gene profiling of parasites remains a difficult task. For example, transcriptome analysis cannot currently be applied to the hepatic stages of the malaria parasite Plasmodium falciparum due to difficulties in obtaining sufficient amounts of parasite material that lies among the large excess of host cell RNA. Here, we describe the isolation of P. falciparum-infected human hepatocytes by a laser capture microdissection approach. Reverse transcriptase polymerase chain reaction amplification of several P. falciparum transcripts demonstrates the high quality of the RNA recovered after microdissection. This approach should enable analysis of P. falciparum transcriptome during its hepatic development, a major step toward the identification of new therapeutic and vaccine targets.

Animals↗
PubMed2005

SRprises along a messenger's journey.

SR (serine/arginine-rich) proteins were originally identified as essential splicing factors. Recent work, however, has implicated these proteins in numerous additional steps of mRNA metabolism, including nuclear export, RNA stability, mRNA quality control, and translation.

Animals↗
PubMed1996

Very fast identification of RNA motifs in genomic DNA. Application to tRNA search in the yeast genome.

A common strategy characterises the various methods independently defined to identify almost unambiguously different types of RNA molecules in DNA fragments. So far, the good quality of detection of RNA motif has been the prior motivation and effectively delayed the optimisation of programs. As an illustration of possible improvements, a modified version of tRNAscan is described. The previous algorithm was altered to run 500 times faster and to lower both rates of false positives and false negatives. The newly sequenced genome of Saccharomyces cerevisiae is scanned both ways in less than three minutes and results match annotations found in databanks with three exceptions, two of which being arguably not real tRNAs.

Algorithms↗
PubMed2006

A highly standardized, robust, and cost-effective method for genome-wide transcriptome analysis of peripheral blood applicable to large-scale clinical trials.

The use of peripheral blood mononuclear cells (PBMC) for transcriptome analysis has already been proven valuable for assessing disease-associated and drug-response-related gene signatures. While these proof-of-principle studies have been critically important, the instability of RNA within PBMC prohibits their use in large-scale multicenter trials for which samples have to be transported for a prolonged time prior to RNA isolation. Therefore, a prerequisite for transcriptome analysis of peripheral blood in clinical trials will be a standardized and valid method to stabilize the RNA profile immediately after blood withdrawal. Moreover, to be able to perform such large-scale clinical studies routinely in several hundred patients more cost-effective array technologies are required. To address these critical issues, we have combined a whole-blood RNA stabilization technology with a method to reduce globin mRNA, followed by genome-wide transcriptome analysis using a newly introduced BeadChip oligonucleotide technology. We demonstrate that the globin mRNA reduction method results in significantly improved data quality of stabilized RNA samples with low intragroup variance and a detection rate of expressed genes similar to that in PBMC. More important, even small differences in gene expression such as are observed between females and males were detected and sufficient to predict gender in whole-blood samples. We therefore propose the combination of globin mRNA reduction after whole-blood RNA stabilization with a newly introduced cost-effective BeadChip array as the preferred approach for large-scale multicenter trials, especially when establishing predictive markers for disease and treatment outcome in peripheral blood.

Blood Cells↗
PubMed2005

Retroposition of processed pseudogenes: the impact of RNA stability and translational control.

Human processed pseudogenes are copies of cellular RNAs reverse transcribed and inserted into the nuclear genome by the enzymatic machinery of L1 (LINE1) non-LTR retrotransposons. Although it is generally accepted that germline expression is crucial for the heritable retroposition of cellular mRNAs, little is known about the influences of RNA stability, mRNA quality control and compartmentalization of translation on the retroposition of processed pseudogenes. We found that frequently retroposed human mRNAs are derived from stable transcripts with translation-competent functional reading frames that are resistant to nonsense-mediated RNA decay. They are preferentially translated on free cytoplasmic ribosomes and encode soluble proteins. Our results indicate that interactions between mRNAs and L1 proteins seem to occur at free cytoplasmic ribosomes.

Animals↗