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At least 37 records · Page 2Linked to original sources
PubMed2006

Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins.

In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion protein comprised of the capsid and nucleocapsid domains of Gag (CANC) and its N-terminally modified mutant (DeltaProCANC) were used to mimic the assembly of the viral core and immature particles, respectively. In contrast to HIV-1, where CANC assembled efficiently into cylindrical structures, the same domains of M-PMV were assembly incompetent. The addition of RNA or oligonucleotides did not complement this defect. In contrast, the M-PMV DeltaProCANC molecule was able to assemble into spherical particles, while that of HIV-1 formed both spheres and cylinders. For M-PMV, the addition of purified RNA increased the efficiency with which DeltaProCANC formed spherical particles both in terms of the overall amount and the numbers of completed spheres. The amount of RNA incorporated was determined, and for both rRNA and MS2-RNA, quantities similar to that of genomic RNA were encapsidated. Oligonucleotides also stimulated assembly; however, they were incorporated into DeltaProCANC spherical particles in trace amounts that could not serve as a stoichiometric structural component for assembly. Thus, oligonucleotides may, through a transient interaction, induce conformational changes that facilitate assembly, while longer RNAs appear to facilitate the complete assembly of spherical particles.

Animals↗
PubMed1996

RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation.

Large quantities of RNA for study by NMR and X-ray crystallography can be produced by transcription reactions in vitro using T7 bacteriophage RNA polymerase. A limitation on producing RNA with this polymerase has been the strong dependence of the yield of the transcription reaction on the sequence at the 5' end of the RNA produced. We report a procedure for obtaining large quantities of enzymatically synthesized RNA from T7 RNA polymerase that has no dependence on the 5' end sequence of the target RNA. Ribonuclease H has been shown previously (Inoue H, Hayase Y, Iwai S, Ohtsuka E, 1987, FEBS Lett 215:327-330) to cleave RNA site specifically using 2'-O-methyl RNA/DNA chimeras to direct the cleavage site. We show that 2'-O-methyl RNA nucleotides on the 5'-side of the DNA nucleotides in the chimera are not essential for site-specific cleavage. This allowed us to design the method such that the same 2'-O-methyl chimera may be used to process any RNA sequence. We have adapted this reaction to the cleavage of NMR-scale quantities of RNA at high yield. RNA is synthesized using T7 RNA polymerase with a 15-nt high-yielding leader sequence at the 5' end, and then this sequence is cleaved off with the RNase H cleavage reaction. The cleaved RNA has 3'-hydroxyl and 5'-phosphate ends, so that the products can be used directly as substrates for ligation by T4 DNA ligase. We show that the cleavage reaction occurs efficiently in solution and on a solid streptavidin/agarose matrix. We report an example in which we are able to improve transcription yield by more than five-fold using this technique in the synthesis of a 15N isotopically labeled hairpin found in the Crithidia fasciculata spliced leader RNA. We are able to obtain a 0.5-mM NMR sample from this inherently poorly transcribing sequence, while minimizing the amount of isotopically labeled rNTPs used to produce it. The NMR spectroscopic results are consistent with the predicted RNA secondary structure.

Animals↗
PubMed2002

Postmortem stability of RNA isolated from bovine reproductive tissues.

Molecular biology is being increasingly used to address the complex problem of bovine infertility. One common concern shared by many of these studies is the postmortem delay in obtaining reproductive tissues and the effect this may have on RNA dependent studies. To address this concern, bovine ovarian, oviduct and uterine tissue samples, collected over intervals ranging from 0 to 96 h postmortem to freeze storage, were analysed to determine the potential effects on RNA quantity and quality. The analysis showed that total RNA yields were not changed significantly by postmortem interval up to 96 h while 28S ribosomal RNA remained intact up to 24 h postmortem. Specific messenger RNA transcripts encoding beta-actin, GAPDH and transforming growth factor-beta were detected in all tissues up to 96 h postmortem using reverse transcriptase-polymerase chain reaction and Northern analysis indicated no detectable mRNA degradation up to 24 h postmortem. Finally, using poly(A)(+) mRNA isolated from ovarian tissues frozen 2 h postmortem, we constructed corpus luteum and ovarian cortex cDNA libraries containing 7.65x10(4) and 1.9x10(6) primary transformants with average cDNA lengths of 2.3 and 1.6 kb respectively. Taken together, these data show that a postmortem delay of up to 24 h does not significantly affect the yield or quality of RNA prepared from bovine reproductive tissues.

Actins↗
PubMed2002

A reverse transcription comparative real-time PCR method for quantitative detection of angiogenic growth factors in head and neck cancer patients.

OBJECTIVES: Head and neck cancer is one of the ten most frequent cancers in the world. The angiogenic growth factors VEGF, PDGF and bFGF play a role in cancer aggressiveness. We developed a sensitive method to quantify the gene expression of these factors in the tissues of head and neck cancer patients. DESIGN AND METHODS: All assays were performed using real-time RT-PCR, which yields a value (Ct) denoting the threshold cycle of PCR amplification at which product is first detected by fluorescence. The Ct is dependent on the quantity of the target molecule in the sample. To control for variation in RNA quantity and quality, we used 18S ribosome RNA as an internal control to calculate a relative Ct for the target molecules of interest, VEGF, PDGF and bFGF. A serially diluted positive control sample was analyzed by linear regression to determine the sensitivity and linearity of the assay. Paired normal and cancerous tissue samples from 115 head and neck cancer patients were assayed to ascertain the relative levels of the growth factors. RESULTS: The CVs of within-run and between-run assays for VEGF, PDGF and bFGF were all less than 3%. The correlation coefficient of the RNA concentrations and Ct values were 0.9987, 0.9977, and 0.9996 respectively for VEGF, PDGF and bFGF. The assay was sensitive to as little as 10(-3) ng of RNA. All three growth factors were significantly increased in tumor tissue as compared to normal tissue. VEGF, PDGF and bFGF levels were elevated in 71.3%, 58.2% and 54.0% of cancerous tissue samples, with average levels of over-expression of 35.1, 24.6 and 13. sixfold, respectively. CONCLUSION: This method provides sensitive, quantitative, high-throughput analysis for direct comparison of gene expression levels between samples, while adjusting for factors that may influence quantity determination. It should be applicable to molecules other than angiogenic growth factors, as well.

Endothelial Growth Factors↗
PubMed2026

Optimized Hot Phenol-Based RNA Extraction from Mycobacteria: A Robust Approach for Reliable Gene Expression Analysis.

Mycobacterium tuberculosis (Mtb) remains a major global health threat, underscoring the need for reliable transcriptomic studies to understand its biology and drug resistance mechanisms. Such analyses depend on obtaining high-quality, high-yield RNA. Although several RNA extraction methods are available, many require expensive reagents, large culture volumes, or specialized equipment, limiting their suitability for large-scale studies, particularly in resource-constrained settings. Here, an optimized Hot Phenol based RNA extraction method specifically tailored for mycobacteria is presented. The method uses minimal culture volume and commonly available reagents to consistently yield high-quality RNA suitable for high-throughput transcriptomic applications. RNA quantity and integrity were assessed by gel electrophoresis and RNA integrity analysis (RIN), and its suitability for downstream applications was confirmed by qPCR and Qubit 4. To benchmark the performance of the optimized method, a parallel RNA extraction using TRIzol and RNeasy under identical experimental conditions was carried out, including the same Mycobacterium species, culture volume, growth phase (logarithmic and stationary), and lysis conditions. This allowed a direct comparison of yield, quality, feasibility, and cost. The optimized Hot Phenol method demonstrated comparable or improved RNA yield and quality while significantly reducing reagent cost and dependence on specialized equipment. Owing to its efficiency, reproducibility, and affordability, this protocol provides a practical alternative for large-scale gene expression and transcriptomic studies in Mtb and other mycobacterial species.

RNA, Bacterial↗
PubMed2005

Macrodissection versus microdissection of rectal carcinoma: minor influence of stroma cells to tumor cell gene expression profiles.

BACKGROUND: The molecular determinants of carcinogenesis, tumor progression and patient prognosis can be deduced from simultaneous comparison of thousands of genes by microarray analysis. However, the presence of stroma cells in surgically excised carcinoma tissues might obscure the tumor cell-specific gene expression profiles of these samples. To circumvent this complication, laser microdissection can be performed to separate tumor epithelium from the surrounding stroma and healthy tissue. In this report, we compared RNAs isolated from macrodissected, of which only surrounding healthy tissue had been removed, and microdissected rectal carcinoma samples by microarray analysis in order to determine the most reliable approach to detect the expression of tumor cell-derived genes by microarray analysis. RESULTS: As microdissection yielded low tissue and RNA quantities, extra rounds of mRNA amplification were necessary to obtain sufficient RNA for microarray experiments. These second rounds of amplification influenced the gene expression profiles. Moreover, the presence of stroma cells in macrodissected samples had a minor contribution to the tumor cell gene expression profiles, which can be explained by the observation that more RNA is extracted from tumor epithelial cells than from stroma. CONCLUSION: These data demonstrate that the more convenient procedure of macrodissection can be adequately used and yields reliable data regarding the identification of tumor cell-specific gene expression profiles.

Carcinoma↗
PubMed2001

Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes.

Genomic DNA contamination within RNA samples has important implications for RT-PCR, particularly if there is a pseudogene related to the gene under investigation, because amplification from pseudogenes and reverse-transcribed cDNA can be very difficult to distinguish. Methods to remove DNA contamination cannot guarantee the absolute absence of DNA from the sample without a loss of RNA quantity or quality, which can be crucial for small amounts of RNA or for the investigation of transcripts with a low level of expression. Here, we describe a general technique for RT-PCR that applies a sequence to the 5' tail of reverse-transcribed cDNA that is not present in genomic DNA and uses this for annealing the reverse PCR primer to exclude genomic DNA amplification in unmodified RNA samples.

Base Sequence↗
PubMed1995

Analysis of transfer RNA during the early embryogenesis of the freshwater teleost, Heteropneustes fossilis.

Total RNA as well as transfer RNA were quantified from mature ova apart from four different embryonic stages namely mid-cleavage, early gastrula, mid-gastrula and organogenesis of the freshwater teleost Heteropneustes fossilis. Total RNA as well as transfer RNA quantity follow a similar variation pattern, being maximum during mid-gastrulation. When analysed by total amino acid acceptance capacity, transfer RNA shows its maximum activity during mid-gastrulation. This coincides with the higher ratio of tRNA to total RNA at this stage. The relative aminoacylation capacity for Ser, Gly, Asn and Thr are found to be higher (9-34%) compared to that for other amino acids. Total tRNA, resolved into three peaks upon HPLC fractionation, shows a high cumulative peak area during mid-gastrulation and organogenesis. These results indicate a switch over of maternal to embryonic translation machinery during gastrulation.

Animals↗
PubMed1976

[Method of localization and evaluation of DNA quantities in bull spermatozoa].

The author attempted to evaluate the DNA quantities by means of a spectrophotometric method of UV light absorption. In a first publication data have been reported on the RNA quantities obtained by this method. This one concerns the data obtained on DNA. By this method, the manipulations on the spermatozoon are more dimple and sources of error are so discarded. It was so possible to get individual data on each spermatozoon, what is possible with chimical methods which give global quantities for a whole sample of semen. It is moreover possible to delimite the localization of DNA and to measure the amounts in each localization. The scale of the obtained data is large enough for information to several workers. The founded quantities and variations in data will be discussed in a following paper.

Animals↗
PubMed2004

Tissue preparation for gene expression profiling of colorectal carcinoma: three alternatives to laser microdissection with preamplification.

Colorectal-carcinoma specimens are heterogeneous and include areas of nonmalignant mucosal and connective tissue. For those study designs in which laser microdissection and RNA preamplification are impracticable, the optimal yield of genuine cancer RNA is a key factor in gene-expression analysis. In this study we compared alternative methods of tissue purification. Three contiguous 0.5-cm(3) samples taken from an advanced primary adenocarcinoma of the sigmoid colon were processed immediately after surgery with the use of the following methods: (1) cryotomy after manual dissection (CMD), (2) microscopically assisted manual dissection (MAMD), and (3) tumor-cell isolation with the use of Ber-EP4 antibodies and Dynabeads (Dynal Biotech GmbH, Hamburg, Germany; technique abbreviated as DB). We generated gene-expression profiles with the use of GeneChip technology (Affymetrix, Santa Clara, Calif) and recorded preparation times, costs, and RNA quantity and quality. CMD took 60 minutes, MAMD 180 minutes, and DB 90 minutes to isolate 22, 8, and 23 microg of RNA, respectively. Expenses for materials amounted to 41, 23, and 91 US dollars for CMD, MAMD, and DB, respectively. The 3'/5' ratio, as determined with the GeneChips, for GAPDH/beta-actin was 1.01:1.03 for CMD, 1.13:1.28 for MAMD, 1.43:1.68 for DB, K-ras, APC, smad 2, transforming growth factor-beta, and p53 were marked as present in all cases, with the exception of APC, which was graded as marginal on DB. The correlation values of gene-expression profiles were 91% (CMD/DB), 93% (CMD/MAMD), and 97% (DB/MAMD). All 3 methods provided enough RNA, of sufficient quality, for gene-expression microarray analysis in colorectal carcinoma. Cross-methodologic analyses of array data should not be performed uncritically.

Adenocarcinoma↗
PubMed2006

RNA integrity and the effect on the real-time qRT-PCR performance.

The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Working with low-quality RNA may strongly compromise the experimental results of downstream applications which are often labour-intensive, time-consuming, and highly expensive. Using intact RNA is a key element for the successful application of modern molecular biological methods, like qRT-PCR or micro-array analysis. To verify RNA quality nowadays commercially available automated capillary-electrophoresis systems are available which are on the way to become the standard in RNA quality assessment. Profiles generated yield information on RNA concentration, allow a visual inspection of RNA integrity, and generate approximated ratios between the mass of ribosomal sub-units. In this review, the importance of RNA quality for the qRT-PCR was analyzed by determining the RNA quality of different bovine tissues and cell culture. Independent analysis systems are described and compared (OD measurement, NanoDrop, Bioanalyzer 2100 and Experion). Advantage and disadvantages of RNA quantity and quality assessment are shown in performed applications of various tissues and cell cultures. Further the comparison and correlation between the total RNA integrity on PCR performance as well as on PCR efficiency is described. On the basis of the derived results we can argue that qRT-PCR performance is affected by the RNA integrity and PCR efficiency in general is not affected by the RNA integrity. We can recommend a RIN higher than five as good total RNA quality and higher than eight as perfect total RNA for downstream application.

Animals↗
PubMed2005

Transcriptional profiling of tissue plasticity: role of shifts in gene expression and technical limitations.

Reprogramming of gene expression has been recognized as a main instructive modality for the adjustments of tissues to various kinds of stress. The recent application of gene expression profiling has provided a powerful tool to elucidate the molecular pathways underlying such tissue remodeling. However, the biological interpretations of expression profiling results critically depend on normalization of transcript signals to mRNA standards before statistical evaluation. A hypothesis is proposed whereby the "fluctuating nature" of gene expression represents an inherent limitation of the test system used to quantify RNA levels. Misinterpretation of gene expression data occurs when RNA quantities are normalized to a subset of mRNAs that are subject to strong regulation. The contention of contradictory biological outcomes using different RNA-normalization schemes is demonstrated in two models of skeletal muscle plasticity with data from custom-designed microarrays and biochemical and ultrastructural evidence for correspondingly altered RNA content and nucleolar activity. The prevalence of these biological constraints is underlined by a literature survey in different models of tissue plasticity with emphasis on the unique malleability of skeletal muscle. Finally, recommendations on the optimal experimental layout are given to control biological and technical variability in microarray and RT-PCR studies. It is proposed to approach normalization of transcript signals by measuring total RNA and DNA content per sample weight and by correcting for concurrently estimated endogenous standards such as major ribosomal RNAs and spiked RNA and DNA species. This allows for later conversion to diverse tissue-relevant references and should improve the physiological interpretations of phenotypic plasticity.

Adaptation, Physiological↗
PubMed1996

Insulin regulation of multiple ribonucleic acid species in human skeletal muscle in insulin-sensitive and insulin-resistant subjects.

In vivo short term (2 h) insulin-regulated gene expression was examined in skeletal muscle of persons with differing insulin sensitivities. Nine genes were analyzed by a S1 nuclease protection assay with multiple probes (multiple S1 nuclease protection assay) to allow the simultaneous examination of RNA abundances from the multiple genes. In insulin-sensitive individuals, 5 of these 9 genes were insulin responsive. RNA from the proto-oncogenes c-Ha-ras, c-myc, and c-src transiently increased 2- to 4-fold within 30 min of insulin infusion. In addition, the RNA abundance of myf-5, a muscle specific differentiation factor, increased 3-fold with a time course similar to that of c-Ha-ras, c-myc, and c-src. In contrast, type 1 protein phosphatase alpha (PPP1A) RNA levels decreased by 50% within 30 min. In insulin-resistant individuals, the RNA levels of c-Ha-ras and myf-5 did not increase, whereas c-src RNA did increase within 30 min of insulin infusion. RNA encoding c-myc transiently increased in both groups; however, this response was lower in insulin-resistant individuals than in insulin-sensitive individuals in a pattern similar to c-Ha-ras and myf-5. PPP1A RNA levels slightly increased in insulin-resistant individuals. In both insulin-sensitive and insulin-resistant persons, RNA quantities of GLUT4, c-jun, c-fos, and the insulin receptor did not change over the period of insulin infusion. However, overall RNA levels of the insulin receptor and c-jun were lower in insulin-resistant individuals.

Adolescent↗