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PubMed2000

Applications of differential-display reverse transcription-PCR to molecular pathogenesis and medical mycology.

The host-fungus interaction is characterized by changes in gene expression in both host and pathogen. Differential-display reverse transcription PCR (DDRT-PCR) is a PCR-based method that allows extensive analysis of gene expression among several cell populations. Several limitations and drawbacks to this procedure have now been addressed, including the large number of false-positive results and the difficulty in confirming differential expression. Modifications that simplify the reaction time, allow the use of minute quantities of RNA, or address unusual species- or gene-specific sequences have been reported. DDRT-PCR has been used to address biological questions in mammalian systems, including cell differentiation, cell activation, cell stress, and identification of drug targets. In microbial pathogenesis and plant pathogenesis, DDRT-PCR has allowed the identification of virulence factors, genes involved in cell death, and signaling genes. In Candida albicans, DDRT-PCR studies identified TIF-2, which may play a role in the upregulation of phospholipases, and the stress-related genes, CIP1 and CIP2. In Histoplasma capsulatum and C. albicans, genes involved in the host-pathogen interaction, including a member of the 100-kDa family in Histoplasma and an ALS and 14-3-3 gene in Candida, were potentially identified by DDRT-PCR. Although very few reports have been published in medical mycology, studies in mammalian, nonfungal microbial, and plant pathogen systems are easily applied to basic questions in fungal pathogenesis and antifungal therapeutics.

Animals↗

PubMed2004

Standardization of preanalytical factors for minimal residual disease analysis in chronic myelogenous leukemia.

Optimal sample quality is a prerequisite to generate valid data in the detection of minimal residual disease (MRD) in leukemias. Thus, the risk of obtaining 'false'-negative results is increased when both quality and quantity of RNA are suboptimal. Factors which affect the sensitivity and consequently the validity of MRD results are reviewed. RNA degradation in unstabilized peripheral blood (PB) samples does not play a major role in samples being processed on the day of blood collection. However, the simulation of sample shipping at room temperature with a delay of sample processing of up to 3 days causes a dramatic loss of intact RNA. RNA degradation can be prevented by the use of a bedside RNA stabilization system. Additionally, the stabilizing procedure is capable of keeping real-time quantitative polymerase chain reaction (RQ-PCR) results comparable whether the sample is processed immediately or with a delay of up to 3 days. Consistent quantitative data cannot be obtained with unstabilized blood samples. Furthermore, the optimum volume of PB required for MRD diagnostics in patients with BCR-ABL-positive chronic myelogenous leukemia in complete cytogenetic remission is revisited. Ten milliliters of PB is sufficient for processing on the day of blood collection whereas the use of only 5 ml PB may result in false-negative results. Standardization of preanalytical and analytical factors is necessary to provide a comparability of RQ-PCR results from different laboratories within multicenter studies. The definition of 'undetectable BCR-ABL' in an individual patient should take these preanalytical parameters into consideration.

Fusion Proteins, bcr-abl↗

PubMed2006

Transcriptome amplification methods in gene expression profiling.

The increasing use of microarray expression profiling to study the molecular biology of cancer and the cellular physiology of difficult-to-isolate cell types has led to a need for methods that accurately and precisely amplify small quantities of RNA. The purpose of this review is to provide an overview of the existing methods for transcriptome amplification and to define the parameters for comparing different amplification methods. The authors propose a standardized protocol for the assessment and evaluation of amplification methods, focusing on a new whole-transcriptome amplification kit, which amplifies total RNA into cDNA fragments. Reproducibility and reliability of the method were analyzed and discussed using both quantitative real-time PCR and a high-density oligonucleotide microarray platform.

DNA, Complementary↗

PubMed1999

Regulation of the expression of NADP-malic enzyme by UV-B, red and far-red light in maize seedlings.

The induction of nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) in etiolated maize (Zea mays) seedlings by UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400-700 nm) was investigated by measuring changes in activity, protein quantity and RNA levels as a function of intensity and duration of exposure to the different radiations. Under low levels of PAR, exposure to UV-B radiation but not UV-A radiation for 6 to 24 h caused a marked increase in the enzyme levels similar to that observed under high PAR in the absence of UV-B. UV-B treatment of green leaves following a 12-h dark period also caused an increase in NADP-ME expression. Exposure to UV-B radiation for only 5 min resulted in a rapid increase of the enzyme, followed by a more gradual rise with longer exposure up to 6 h. Low levels of red light for 5 min or 6 h were also effective in inducing NADP-ME activity equivalent to that obtained with UV-B radiation. A 5-min exposure to far-red light following UV-B or red light treatment reversed the induction of NADP-ME, and this effect could be eliminated by further treatment with UV-B or red light. These results indicate that physiological levels of UV-B radiation can have a positive effect on the induction of this photosynthetic enzyme. The reducing power and pyruvate generated by the activity of NADP-ME may be used for respiration, in cellular repair processes and as substrates for fatty acid synthesis required for membrane repair.

Enzyme Induction↗

PubMed1998

Effect of TGF-beta 1 on PDGF receptors expression in human scar fibroblasts.

This study examined the effect of exogenous TGF -beta1 on platelet derived growth factor alpha and beta (PDGF-alpha, beta) receptor expression in human dermal fibroblasts derived from both normal cutaneous tissues (normal skin [NSk]) and (normal scar [NSc]) and abnormal scar (keloid). TGF-beta and PDGF are present in the early phases of wound healing and are implicated in tissue fibrosis. In this study, replicate samples of NSk, NSc and keloid fibroblasts were grown to subconfluency in DMEM/10% FBS followed by replacement of media with DMEM/0.1%FBS for 24 hrs. One group of cells (NSk, NSc and keloid) were exposed to 10 ng/mL of exogenous TGF-beta1 for 24 hours, while the other group was used as control with no exposure to exogenous TGF-beta1. RadioImmunoBinding assays, Western and Northern blot analysis were performed to examine both PDGF-alpha and PDGF-beta receptor expression at the transcriptional and post-transcriptional levels. cDNA receptor probes were synthesized using polymerase chain reaction (PCR) with selected primer sets derived from published sequences. Beta-actin probe was used as a control to confirm that the same quantity of RNA was used for each experimental condition. TGF-beta1 was found to upregulate the expression of PDGF-alpha receptor for keloid fibroblasts but not for NSk or NSc fibroblasts. No effect was observed for TGF-beta 1 on PDGF-beta receptor expression for any of the cell lines examined.

Blotting, Northern↗

PubMed2005

Rational probe optimization and enhanced detection strategy for microRNAs using microarrays.

MicroRNAs (miRNAs) are post-transcriptional regulators participating in biological processes ranging from differentiation to carcinogenesis. We developed a rational probe design algorithm and a sensitive labelling scheme for optimizing miRNA microarrays. Our microarray contains probes for all validated miRNAs from five species, with the potential for drawing on species conservation to identify novel miRNAs with homologous probes. These methods are useful for high-throughput analysis of micro RNAs from various sources, and allow analysis with limiting quantities of RNA. The system design can also be extended for use on Luminex beads or on 96-well plates in an ELISA-style assay. We optimized hybridization temperatures using sequence variations on 20 of the probes and determined that all probes distinguish wild-type from 2 nt mutations, and most probes distinguish a 1 nt mutation, producing good selectivity between closely-related small RNA sequences. Results of tissue comparisons on our microarrays reveal patterns of hybridization that agree with results from Northern blots and other methods.

Animals↗

PubMed2001

Identification of genes expressed in the Xenopus inner ear.

Recent studies indicate that hearing loss in humans has strong hereditary components associated with expression of specific genes in the auditory apparatus of the inner ear. However, the inner ear poses challenges for molecular research because the amount of tissue that can be isolated is limited, and extraction procedures yield small quantities of RNA and protein. To begin to identity genes essential for auditory function, we synthesized a cDNA library using an RT-PCR protocol and total RNA isolated from eight Xenopus laevis inner ears. Sequence analysis of randomly selected clones demonstrated expression of both identified (calmodulin, SNARE protein, syndecan-2) and unidentified genes, and confirmed synthesis of full length transcripts. Confocal and scanning electron microscopy (SEM) were used to examine the structure of inner ear organs that serve as auditory receptors in amphibians: the sacculus, the amphibian papilla and the basilar papilla. SEM images illustrate the heterogeneity of bundle morphology and demonstrate the continuous appearance of stereociliary bundles in the X. laevis amphibian papilla during larval development and adult life. Investigations of gene expression in Xenopus auditory organs using clones recovered from inner ear cDNA libraries should provide insight regarding the molecular basis of hearing.

Animals↗

PubMedDate not supplied

Expression of the myc protooncogene in canine rhabdomyosarcoma.

The expression of the protooncogene c-myc in a canine rhabdomyosarcoma was examined. It was found that this highly malignant tumour contained vast quantities of RNA that hybridized with a cDNA probe for c-myc. Restriction fragment length analysis after endonuclease digestion of tumour DNA did not reveal any rearrangements in this gene locus. The potential role of this oncogene in the development of canine rhabdomyosarcoma is discussed.

Animals↗

PubMed1994

Quantitative analysis of folylpolyglutamate synthetase gene expression in tumor tissues by the polymerase chain reaction: marked variation of expression among leukemia patients.

Evidence from previous in vitro studies indicates that the enzyme folylpolyglutamate synthetase (FPGS) may be an important determinant of the antitumor activity of antifolate drugs that are substrates for this enzyme. To facilitate investigations regarding the association between FPGS content of tumor tissues and the sensitivity of tumors to antifolates, we developed a polymerase chain reaction (PCR)-based gene expression quantitation assay for measuring relative amounts of FPGS mRNA in tumor tissue specimens. From the known sequence of the human gene, FPGS-specific PCR primers were chosen that flanked a 263-base segment of the FPGS gene. The PCR carried out with these primers was linear over at least a three orders of magnitude range of starting cDNA concentration. The amount of cDNA required per assay corresponded to the quantity of RNA contained in nanogram to microgram amounts of tissue, depending on the level of gene expression. In CHO AUXB1 (FPGS) cell lines transfected with human DNA and expressing different levels of human FPGS, FPGS gene expression measured by this assay was linear with the FPGS enzyme activity in the cells. In human head and neck cell lines, which contained naturally varying levels of FPGS enzyme activity, FPGS gene expressions were also linearly proportional to FPGS enzyme content as measured both by activity in cell-free extracts and by intracellular methotrexate polyglutamate formation. Among leukemic cells from 11 acute lymphocytic leukemia and acute myelogenous leukemia patients, FPGS expression varied by over 500-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1993

Identification of alpha 1C-adrenergic receptor mRNA in bovine retinal pigment epithelium.

PURPOSE: To examine the localization of a novel alpha 1-adrenergic receptor subtype of alpha 1C receptor in the eye, we compared the amount of alpha 1C receptor transcripts in bovine retinal pigment epithelium (RPE) and in neural retina by employing reverse transcription of mRNA and the polymerase chain reaction (RT-PCR) assay. METHODS: RT-PCR assay with specific primers for the alpha 1C-adrenergic receptor and for beta-actin showed linear relationships between input quantity of RNA and the amount of amplified PCR products for the alpha 1C-adrenergic receptor and also for beta-actin, when PCR was conducted for 24 and 15 cycles, respectively. RESULTS: The RT-PCR assay demonstrated that the spontaneous expression level of the alpha 1C-adrenergic receptor mRNA was much higher in bovine RPE than in neural retina; the alpha 1C-adrenergic receptor/beta-actin ratio from RPE was 0.33 +/- 0.15 (n = 4), whereas that from neural retina was virtually zero. CONCLUSIONS: The RT-PCR assay is a sensitive semiquantitative method for a low abundance mRNA in a limited number of cells. Using the alpha 1C receptor as a model, we demonstrated the usefulness of this assay by showing the uneven distribution of the alpha 1C receptor transcripts in bovine RPE cells and neural retina.

Actins↗

PubMed2005

Classification of human tumors using gene expression profiles obtained after microarray analysis of fine-needle aspiration biopsy samples.

BACKGROUND: Gene expression profiling using gene-discovery, high-density microarray technologies is a powerful tool. One potential application is the development of tumor classifiers that predict the site of origin. For this technology to be relevant, however, it must be applicable to tumor biopsy samples, which most often are fine-needle aspiration biopsy (FNAB) samples. METHODS: Surgically resected tumors were sampled by FNAB using different gauge needles. A portion of the excised tumor was also collected. RNA samples were extracted using standard techniques and the quality and quantity of the RNA samples were measured for each sample. Thirteen representative FNAB samples and two representative tissue samples were submitted for microarray analysis and then subjected to a tumor classifier. RESULTS: Fourteen of 18 samples analyzed for quantity and quality of RNA yielded an adequate amount of RNA (> 1 microg total RNA). Tumor type contributed to the RNA yield because one of the four inadequate samples was retrieved from a patient with lobular carcinoma of the breast and the other three samples were retrieved from patients with retroperitoneal sarcomas. Of the 13 samples submitted for microarray analysis, 9 were classified correctly as to tumor type using a tissue-based tumor classifier. CONCLUSIONS: The authors demonstrated that FNABs reproducibly obtained an adequate amount of RNA for microarray analysis when a standardized collection procedure was used. Furthermore, the samples generated interpretable gene expression profiles that could be matched accurately with a tumor classifier established on tissue specimens. The current study showed that FNAB produced adequate material for microarray analysis when utilizing a standardized collection procedure.

Biopsy, Fine-Needle↗

PubMed1996

Hepatitis C virus infection associated with administration of intravenous immune globulin. A cohort study.

OBJECTIVE: To determine the risk of and risk factors for hepatitis C virus (HCV) infection among persons with immune deficiencies who had received intravenous immune globulin (IGIV) between March 1993 and February 1994. DESIGN: Retrospective cohort study. SETTING: An immunology program in a tertiary care hospital. PATIENTS: Of 341 persons who had received IGIV between March 1, 1993, and February 22, 1994, 278 (82%) were enrolled. The mean age for the enrolled persons was 9 years, and 99% had primary immune deficiencies. MAIN OUTCOME MEASURES: Evidence of HCV infection by detection in sera of antibody to HCV and/or HCV RNA by reverse transcriptase polymerase chain reaction. RESULTS: Twenty-three (11%) of 210 persons who received the IGIV Gammagard (Baxter Healthcare Corporation, Deerfield, Ill) became infected compared with none of 52 persons who received exclusively other IGIV products (P=.01). In a multivariate analysis, HCV infection was associated only with Gammagard produced from plasma screened by second-generation (multiantigen) anti-HCV tests (P=.03). Hepatitis C virus RNA was detected in Gammagard, and the risk of transmission to recipients increased with increasing quantity of HCV RNA infused, from 0 for those who received no HCV RNA-positive lots to 29% for the quartile of patients receiving the greatest amount (P<.001). At least 9 different lots of Gammagard were required to account for all cases. CONCLUSION: Gammagard was the only IGIV product implicated in the transmission of HCV. Infection was associated with higher quantities of HCV RNA in Gammagard produced from second-generation anti-HCV-screened plasma. Further studies are needed to determine reasons for the infectivity of Gammagard, and viral inactivation and removal steps are needed to ensure the safety of IGIV products.

Alanine Transaminase↗

PubMed2006

RNA quality and gene expression analysis of ovarian tumor tissue undergoing repeated thaw-freezing.

Gene expression profiles evaluated by microarray-based quantization of RNA are used in studies of differential diagnosis and prognosis in cancer. RNA of good quality is mandatory for this evaluation. The RNA most often comes from tumor banks with limited amount of tissue, and the tissue often undergoes repeated thawing and freezing. We evaluated the influence of repeated division of tumor samples at room temperature, on RNA quality and quantity, in addition to the gene expression profile. Sixteen ovarian tumor samples were divided in three aliquots each, undergoing respectively one, two, and three thaw-freeze cycles. RNA from each aliquot was extracted on the day of division, and quantity and quality were evaluated. RNA from all three aliquots of four tumor samples underwent microarray analysis on Affymetrix Human Genome U133A 2.0 arrays. Microarray data were evaluated using both unsupervised, and supervised multivariate statistical methods, reliability analysis, as well as verification using published gene lists in ovarian cancer studies. RNA quality and quantity did not change during the division procedure and microarray data showed insignificant difference in gene expression. Tumor samples from tumor banks can be frozen and thawed at least three times without compromising the RNA integrity and genetic expression profile.

Cryopreservation↗

PubMed2003

RNA crystallization.

RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to synthesize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

Base Sequence↗

PubMed2004

[Progress towards the use of DNA microarray technology for the study of wild Plasmodium strains].

Plasmodium falciparum is still a major cause of mortality in the world. Due to growing drug resistance in most endemic countries, the use of tools allowing large-scale analysis of P. falciparum biology is increasingly urgent. In addition to gene sequence data, post-genomic methods including microarray-based transcript profiling allow complete Plasmodium gene expression. However, application of this technology has been limited to study of samples presenting large quantities of total RNA (8 microg to 50 microg). Indeed at least two replicas (one technical and one biological) are necessary to ensure the statistical strength of results. This constraint excludes the use of biological materials hardly available and of wild strain samples. Many methods have been developed to facilitate the use of microarray technology with smaller quantities of total RNA. Currently the use of amplification techniques, various fluorochrome markers, and labelled cDNA and/or RNA probes avoiding all amplification steps, to enhance detection sensitivity has enabled reliable assays to be performed with less material. However these enhancement techniques appear to have a biasing effect on results and the use of powerful new markers is still limited for technical and practical reasons. At the present time radioactive labeling is the most reliable technique for assays using small quantities of total RNA. This approach is not only compatible with competitive hybridization but also enables all microarray screening criteria. Findings from our laboratory support the effectiveness of radioactive labeling for microarray-based determinations on P. falciparum.

Animals↗

PubMed1961

Studies of E. coli ribosomal RNA and its degradation products.

The RNA of E. coli ribosomes has been extracted by the phenol method. The 70S ribosomes contain RNA molecules of 28 and 18S almost exclusively. When the 70S ribosomes are dissociated to 30 and 50S ribosomes the former contain only the 18S RNA and the latter a mixture of 28 and 18S RNA. There are also present, however, small quantities of ribosomal RNA having sedimentation coefficients of between 4 and 8S. These small molecules are particularly abundant in the smaller ribosomes present in the cell extract and account for most of the RNA of 20S ribosomes.In addition it has proved possible to degrade the large molecules of RNA to a series of smaller molecules. Removal of magnesium ions from the growing cell, extensive dialysis of the RNA against a buffer of low ionic strength, and heating all resulted in such degradation. Three degradation products were observed having sedimentation coefficients of about 13.1S, 8.8S, and 4.4S. The integral sedimentation distributions of these preparations suggest a high degree of homogeneity among the molecules of each of the three classes. The three sizes seem to result from sequential breaks in the molecules since the proportion of smaller molecules increases with time of treatment.The molecular weights of the 8.8S and 4.4S molecules have been estimated as 144,000 +/- 4,900 and 29,200 +/- 1,200 respectively by the Archibald method.

Escherichia coli↗

PubMed2004

Recent approach for diagnosis of early HCV infection.

Detection of HCV infection during the window phase of infection, before seroconversion, is important in blood screening. However, a significant delay exists between the time of infection and the development of antibodies. The delay in window period can last up to 70 days. The aim of the present study was to investigate the kinetics of HCV markers during early infection, with detection of HCV core antigen as an early method for diagnosis. The study included determination of HCV RNA by qualitative and quantitative PCR, HCV core antigen detection by enzyme linked immunosorbent assay (ELISA) and specific serological markers including anti-HCV IgG and IgM. The study was carried out on 34 patients diagnosed as non A non B acute hepatitis and proved to be hepatitis C by qualitative HCV RNA PCR. Sixteen healthy control subjects were also included. From each consenting patient and control, blood samples were collected and serum was separated and subjected to determination of AST and ALT and the following virological laboratory tests: HCV core antigen detection by ELISA, determination of specific anti-HCV IgM and specific anti-HCV IgG, qualitative and quantitative determination of HCV RNA by second version of PCR. In patients, the median quantity of HCV RNA was 739.1 x 10(3) lu/ml with minimum quantity 2.1 x 10(3) lu/ml and maximum 38352.3 x 10(3) lu/ml. A comparison between the different diagnostic methods revealed that the highest sensitivity was for HCV-core antigen detection (82.4%), specificity was 100% negative predictive value was 72.2% and positive predictive value was 100%. Specific anti-HCV IgG had moderate levels of sensitivity (58.5%), specificity (75%), negative predictive value (46.2%)and positive predictive value (83.3%). The least sensitive method was the specific anti-HCV IgM (29.4%) with negative predictive value 40% but had specificity and positive predictive value of 100% of each. From this study we could conclude the followings: From virological methods, serological detection of specific IgM anti-HCV had the least sensitivity limits, while it had the highest specificity and positive predictive value. Specific anti-HCV IgG had moderate sensitivity and specificity. The most sensitive and specific tool for diagnosis of early HCV viraemia was the detection of HCV core Ag by ELISA when compared to molecular biological methods.

Adult↗

PubMed2006

Isolation of high-quality RNA from apple (Malus domestica) fruit.

It is difficult to isolate sufficient quantities of high-quality RNA from apple fruit. An abundance of polyphenolic compounds and polysaccharides and a relatively low concentration of RNA in the fruit tissue create conditions that hamper RNA isolation when standard techniques are used. We have developed two RNA isolation methods that include an initial homogenization and extraction with acetone or ethanol. These in turn remove the interfering compounds and precipitate the protein and nucleic acids for subsequent RNA extraction. The quality of RNA was satisfactory with both acetone and ethanol preparations; however, the acetone powder produced consistently higher quantities of RNA.

Acetone↗