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At least 577 records · Page 32Linked to original sources
PubMed2003

The effect of ribavirin and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA.

The recent development of in vitro hepatitis C virus (HCV) RNA replication systems has provided useful tools for studying the intracellular anti-HCV activity of ribavirin. Ribavirin has been shown to: (1) induce "error catastrophe" in poliovirus, Proc. Natl. Acad. Sci. USA 98, 6895-6900), (2) be a pseudo-substrate of the HCV RNA-dependent RNA polymerase (RdRp) in vitro, J. Biol. Chem. 276, 46094-46098), and (3) increase mutations in HCV RNA in the binary T7 polymerase/HCV cDNA replication system, J. Virol. 76, 8505-8517). These findings have led to the hypothesis that ribavirin may also induce error catastrophe in HCV. However, the functional relevance of ribavirin-induced HCV RNA mutagenesis is unclear. By use of a colony formation assay, in which RNA is isolated from the HCV subgenomic replicon system following treatment, the impact of ribavirin, inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors, and the combination was assessed. Ribavirin reduced HCV replicon colony-forming efficiency (CFE) in a dose-dependent fashion, suggesting that ribavirin may be misincorporated into replicon RNA and result in an anti-replicon effect analogous to error catastrophe. This effect was markedly suppressed by addition of exogenous guanosine. Combination treatment with ribavirin and mycophenolic acid (MPA) or VX-497, both potent, nonnucleoside IMPDH inhibitors, led to a greatly enhanced anti-replicon effect. This enhancement was reversed by inclusion of guanosine with the treatment. In contrast, MPA or VX-497 alone had only marginal effects on both the quantity and quality (CFE) of replicon RNA, suggesting that although IMPDH inhibition is an important contributing factor to the overall ribavirin anti-HCV replicon activity, IMPDH inhibition by itself is not sufficient to exert an anti-HCV effect. Sequencing data targeting the neo gene segment of the HCV replicon indicated that ribavirin together with MPA or VX-497 increased the replicon error rate by about two-fold. Taken together these results further suggest that lethal mutagenesis may be an effective anti-HCV strategy. The colony formation assay provides a useful tool for evaluating mutagenic nucleoside analogs for HCV therapy. Finally, the data from combination treatment indicate potential therapeutic value for an enhanced anti-HCV effect when using ribavirin in combination with IMPDH inhibition.

Antimetabolites↗
PubMedDate not supplied

RNA-sequence-mediated gene regulation in HIV-1.

The quantity and quality of human immunodeficiency virus type 1 (HIV-1) gene expression is controlled in large part by the action of two small nuclear viral regulatory proteins termed Tat and Rev. Tat is unique among transcriptional trans-activators in that it acts via a structured RNA target sequence, termed TAR, to induce high levels of transcription from the HIV-1 long terminal repeat promoter element. The activity of the viral Rev protein is also unprecedented in that this protein functions to induce the nuclear export of a specific class of viral RNA species that are otherwise sequestered in the nucleus by the action of cellular factors. Like Tat, Rev also interacts with a highly specific cis-acting target sequence termed, in this case, the Rev Response Element. In this review, I provide an outline of our current understanding of the roles and mechanisms of action of these two novel RNA-sequence-dependent regulatory proteins.

Base Sequence↗
PubMed2004

Detection of tumor-specific mRNA in cell-free bronchial lavage supernatant in patients with lung cancer.

Bronchoscopy is a standard procedure in the workup of patients with suspicious pulmonary lesions. We wondered whether it is possible to isolate malignancy-associated mRNA from cell-free lavage supernatant. Extracellular mRNA from cell-free lavage supernatant of 25 patients with lung cancer (23 with non-small cell lung cancer, 2 with small cell lung cancer) was isolated, reverse-transcribed, and amplified by reverse transcriptase polymerase chain reaction. The quantity and quality of the isolated RNA were checked after cDNA synthesis by amplification with beta-actin-specific primers. Afterwards, a panel of eight genes known to be expressed in lung tumors was used for the detection of tumor-associated mRNA expression in lavage supernatant and serum. mRNA coding for beta-actin could be isolated from lavage supernatant of all 25 patients. In addition, the expression of at least one tumor-associated gene was detectable in all patients. These results show that intact mRNA can be isolated from cell-free lavage supernatant and that its quantity and quality are sufficient for the detection of tumor-associated gene expression alterations. This may open new possibilities for the diagnosis of lung cancer.

Actins↗
PubMed1975

DNA-dependent RNA polymerases I and II from kidney. Effect of polyamines on the in vitro transcription of DNA and chromatin.

DNA-dependent RNA polymerases I and II were purified from pig kidney nuclei by chromatography on DEAE-Sephadex and phosphocellulose. When nonlimiting amounts of double-stranded DNA were used as the template, the in vitro transcription was markedly stimulated by spermidine and spermine. Maximal stimulation of RNA polymerase I occurred at 2-5 mM spermidine and 0.5-2 mM spermine, whereas optimal polyamine concentrations for RNA polymerase II were 5-10 and 1-5 mM for spermidine and spermine, respectively. DNA transcription by polymerase II was stimulated to a greater extent than that of polymerase I. Higher spermine (5-10 mM) concentrations were strong inhibitors of both polymerases under these conditions. The apparent Km of RNA polymerases I and II for UTP was unchanged at optimal polyamine concentration; under the same conditions the maximal reaction velocity was increased two- to three-fold and was essentially due to an increase in the rate of chain elongation. Thus, in a typical experiment the average chain length as determined by the UMP/uridine ratio increased from 570 to 1330 and the chain elongation rate increased from 0.64 to 1.44 nucleotides times sec-1 in the presence of spermine. When limiting quantities of native DNA were employed as the template, both RNA polymerases I and II were inhibited by 1-2 mM spermine. Kidney chromatin could be transcribed by homologous RNA polymerases with an efficiency ranging from 2 to 10% of that with native DNA. When chromatin was used in nonlimiting amounts instead of DNA, RNA polymerase II activity was again stimulated about two-fold at 2 mM spermine. Under these conditions, RNA polymerase I activity was inhibited by spermine. The inhibition of RNA synthesis in vitro at limiting quantities of templates (DNA or chromatin) could be overcome by preincubation of the enzyme with templates before polyamines were added. This inhibition thus appears to be due to a block in the initiation of RNA chains. Similar inhibition of transcription by RNA polymerase II was also observed with limiting quantities of chromatin as the template.

Animals↗
PubMed1993

Construction of overlapping oligonucleotide templates for the production of cRNA standards for quantitative reverse transcription polymerase chain reaction.

The reverse transcriptase polymerase chain reaction (PCR) is a technique for the study of gene expression that requires far less RNA for analysis than Northern blots. The inclusion of cRNA standards in the initial reverse transcription step is a way to control for the tube-to-tube variation often inherent in the technique and to permit quantitation of the starting amount of the native mRNA being analyzed. We describe a method using overlapping oligonucleotides to produce templates for the production of cRNA standards for up to three different mRNA species. The first step is the synthesis of a pair of overlapping oligonucleotides each of which encodes, respectively, sequences analogous to either sense or antisense primers for the PCR amplification of up to three different messages. These oligonucleotides are designed to have complementary 3' ends which permit spontaneous annealing and allow subsequent mutually priming extension of the annealed double-stranded portion by T7 DNA polymerase. The T7 and SP6 RNA polymerase promoters are then added to the ends of the template using standard PCR techniques. Once the template is assembled, T7 and SP6 RNA polymerases are used to produce copious quantities of cRNA standards and controls. This technique can be used to construct multiple cRNA standards for essentially any messages of interest. Production of cRNA by a single T7 RNA polymerase reaction yields standards sufficient for several thousand separate reverse transcriptions.

Base Sequence↗
PubMed1994

Identification of the cis-acting signal for minus-strand RNA synthesis of a murine coronavirus: implications for the role of minus-strand RNA in RNA replication and transcription.

Minus-strand RNA is the first RNA species made by plus-strand RNA viruses, such as mouse hepatitis virus (MHV), and serves as a template for subsequent RNA replication and transcription. The regulation of minus-strand RNA synthesis has been difficult to study because of the paucity of minus-strand RNA. We have optimized a ribonuclease (RNase) protection assay which enabled the detection of minus-strand RNA synthesis from nonreplicating RNAs, thus clearly separating minus-strand from plus-strand RNA synthesis. We used an MHV defective interfering (DI) RNA containing a chloramphenicol acetyltransferase gene as a reporter to determine the cis-acting signal for MHV minus-strand RNA synthesis. It was found that minus-strand RNAs existed in double-stranded RNA form in the cell. By using various deletion clones, we demonstrated that the cis-acting signal for minus-strand RNA synthesis resides in the 55 nucleotides from the 3' end plus poly(A) tail of the MHV genome. This is much shorter than the 436 nucleotides previously reported for the 3'-end replication signal. No specific upstream MHV sequence was required for the initiation of minus-strand RNA synthesis. This finding suggests that the requirement for minus-strand RNA synthesis is much less stringent than that for genomic and subgenomic plus-strand RNA synthesis and that some of the minus-strand RNAs made may not be functional since they may lack the recognition signals for RNA replication or transcription. We further showed that the DI clones which actively transcribed a subgenomic mRNA from an internal intergenic sequence synthesized much less minus-strand RNA than those clones which did not transcribe subgenomic mRNAs, indicating that minus-strand RNA synthesis was inhibited by transcription from an internal promoter of the same DI RNA. This result also suggests that the regulation of the quantities of subgenomic mRNAs is not at the point of minus-strand RNA synthesis but rather at plus-strand RNA synthesis. Furthermore, the finding that the leader sequence was not required for minus-strand RNA synthesis suggests that the leader RNA regulates mRNA transcription during plus-strand RNA synthesis.

Animals↗
PubMed2005

High reproducibility using sodium hydroxide-stripped long oligonucleotide DNA microarrays.

Recently, long oligonucleotide (60- to 70-mer) microarrays for two-color experiments have been developed and are gaining widespread use. In addition, when there is limited availability of mRNA from tissue sources, RNA amplification can and is being used to produce sufficient quantities of cRNA for microarray hybridization. Taking advantage of the selective degradation of RNA under alkaline conditions, we have developed a method to "strip" glass-based oligonucleotide microarrays that use fluorescent RNA in the hybridization, while leaving the DNA oligonucleotide probes intact and usable for a second experiment. Replicate microarray experiments conducted using stripped arrays showed high reproducibility, however, we found that arrays could only be stripped and reused once without compromising data quality. The intraclass correlation (ICC) between a virgin array and a stripped array hybridized with the same sample showed a range of 0.90-0.98, which is comparable to the ICC of two virgin arrays hybridized with the same sample. Using this method, once-stripped oligonucleotide microarrays are usable, reliable, and help to reduce costs.

Breast Neoplasms↗
PubMed1989

Probes for quantitating subpicogram amounts of HIV-1 RNA by molecular hybridization.

A set of probes was designed for the quantitation of HIV-1 RNA in infected cells by a molecular hybridization procedure called reversible target capture. Reversible target capture is analogous to sandwich hybridization, except that the link between hybrid complexes and the affinity support was reversible, allowing for repeated capture of hybrids on, and release from, fresh affinity support. Repeated cycles of capture resulted in a high degree of purification of hybrids from unreacted probe, thereby greatly reducing assay noise and increasing assay sensitivity. Probes against the HIV-1 pol gene were chosen because their target sequences were highly conserved among HIV-1 isolates, while being divergent enough to provide discrimination from other human T cell tropic viruses. Subpicogram quantities of HIV-1 pol gene RNA were measured with signal:noise ratios of over 10. Hybridization signal increased with increasing target RNA with a proportionality constant of 1.

Cells, Cultured↗
PubMed2006

Real-time quantitative analysis for human telomerase reverse transcriptase mRNA and human telomerase RNA component mRNA expressions as markers for clinicopathologic parameters in urinary bladder cancer.

AIM: The expression of the telomerase subunits such as human telomerase reverse transcriptase (hTERT) and human telomerase RNA component (hTR) may be associated with tumor development and progression. We evaluated the relationship between mRNA quantification of both hTERT and hTR and clinicopathologic parameters in bladder cancer. METHODS: We examined the mRNA expression of hTERT and hTR in 29 specimens with bladder cancer (Grade: Grade I, 9 cases; Grade II, 13 cases and Grade III, 7 cases. Stage: pTa-pT1, 18 cases; pT2-T4, 11 cases). We immediately froze all of specimens obtained during TUR-Bt and isolated the total RNA from each specimen. We measured the quantity of hTERT, hTR and GAPDH mRNA by a real-time reverse transcription-polymerase chain reaction method based on TaqMan technology. RESULTS: The hTERT/GAPDH mRNA ratio and hTERT mRNA/total RNA in superficial bladder tumor was significantly lower than in invasive bladder tumor. The hTR/GAPDH mRNA ratio and hTR mRNA/total RNA in superficial tumor were significantly lower than in invasive bladder tumor. The hTERT mRNA expression significantly correlated with tumor grade, but the hTR mRNA expression did not correlate with tumor grade. There was no significant difference in the hTERT/GAPDH mRNA ratio and hTR mRNA/total RNA according to multiplicity of bladder tumor. CONCLUSIONS: Our results demonstrated that the expression of hTERT mRNA correlated with the progression of stage and grade in bladder cancer. The quantitative analysis of hTERT and hTR mRNA might be a marker for clinicopathologic parameters in bladder cancer.

Adult↗
PubMed2004

Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues.

Although RNA can be retrieved from formalin-fixed, paraffin-embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol-fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin-fixed tissues. RNA from ethanol-fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT-PCR using RNA from ethanol-fixed tissues was consistently successful. RNA from FFPE tissues composed of low-molecular weight fragments, and their use in RT-PCR failed repeatedly. The yield and quality of RNA from ethanol-fixed brains were unaffected after immersion at 4 degrees C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol-fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol-fixed tissues for RT-PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.

Animals↗
PubMed1983

Construction of a synthetic messenger RNA encoding a membrane protein.

We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.

Animals↗
PubMed1992

Large scale chemical synthesis, purification and crystallization of RNA-DNA chimeras.

RNA-DNA chimeras, in which both DNA and RNA monomers are site-specifically substituted in the same strand, may be prepared only by chemical synthesis. Biochemical studies have revealed a number of surprising and subtle effects resulting from the insertion of either a ribonucleotide into a DNA strand or a deoxyribonucleotide into an RNA strand. The availability of large quantities of these chimeras allows for their crystallization and subsequent x-ray structure determination. We describe a flexible and efficient method for the large-scale preparation of these compounds, their purification, and their crystallization. The methodology is based on a combination of existing DNA phosphoramidite synthons and those recently introduced for the preparation of biochemically active RNA1. We demonstrate that these two different synthons are compatible, produce large quantities of nucleic acid needed for physical studies, and that high resolution diffraction quality crystals may be grown from these chimeras. Of the duplex chimeras synthesized and crystallized, [r(G)d(CGTATACGC)]2, [d(GCGT)r(A)d(TACGC)]2 and [r(GCG)d(TATACCC) + d(GGGTATACGC)] form A-helices and d(CG)r(CG)d(CG)]2 forms a left-handed Z-helix.

Base Sequence↗
PubMed1970

Preparation, purification and analyses of thirteen alkali-stable dinucleotides from yeast ribonucleic acid.

Of the 16 alkali-stable dinucleotides known to be obtained by hydrolysis of commercial yeast RNA with alkali, 13 were prepared in quantities of the order of 10mg or more. The samples, with only one exception, contain at least 90% of dinucleotide, and spectroscopic constants and nucleotide-sequence determinations, although not conclusive, indicate a high degree of purity of these products. The small dinucleotide fraction in 150g of RNA hydrolysed with alkali (1-2% of the total nucleotides) was separated from the mononucleotides by stepwise ion-exchange chromatography on DEAE-cellulose columns and resolved into seven fractions containing from one to four different dinucleotides by electrophoresis on paper at pH3.0. These fractions were resolved into their constituent dinucleotides by chromatography in ammonium sulphate. Contamination of the products by impurities from the paper was minimized by washing it before using it for chromatography or electrophoresis and, by using a thick grade of paper (Whatman no. 17), it was possible to handle and purify relatively large quantities of nucleotides.

Alkalies↗
PubMed2001

Inhibition of telomerase by site-specific ribonucleases in gastric and esophageal adenocarcinoma.

Increased expression of telomerase is critical in the pathogenesis of cancer. Telomerase expression is reported variably in foregut cancers, possibly as a result of telomerase inhibition or ribonucleases. We performed experiments to assess telomerase and telomerase RNA expression in foregut cancers and to quantify and characterize telomerase inhibition. Cancer specimens were obtained from 27 patients. Telomerase activity of cancers was determined by the telomeric repeat amplification protocol, the presence of telomerase RNA component (hTERC) by reverse transcription PCR, and the quantity of telomerase inhibitors in mixing experiments. Ribonuclease activity was measured by assessing degradation of labeled RNA by cancers. Telomerase was found in 8/11 adenocarcinomas of the esophagus or gastroesophageal junction and 6/16 distal gastric adenocarcinomas; hTERC was detectable in all cancers. Telomerase inhibition was more marked in distal compared to proximal adenocarcinomas (P = 0.01) and correlated with ribonuclease activity (rS = 0.65). Ribonucleases contribute significantly to telomerase inhibitory activity detectable in foregut cancer specimens. In vitro, the presence of telomerase inhibitors in some specimens did not prevent the detection of telomerase by the TRAP assay. This suggests a more complex relationship between telomerase and its inhibitors. Site-specificity of telomerase inhibitors generally and ribonuclease activity specifically suggests a putative regulatory role in vivo.

Adenocarcinoma↗
PubMed1995

The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis.

Cytotoxic T lymphocytes (CTL) are thought to contribute to viral clearance and liver cell injury during hepatitis B virus (HBV) infection. Using a strategy involving the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with HBV-derived synthetic peptides containing HLA-A2.1, -A31, and -Aw68 binding motifs, we have previously described CTL responses to several epitopes within the HBV nucleocapsid and envelope antigens in patients with acute hepatitis. In this study we define six HLA-A2-restricted CTL epitopes located in the highly conserved reverse transcriptase and RNase H domains of the viral polymerase protein, and we show that the CTL response to polymerase is polyclonal, multispecific, and mediated by CD8+ T cells in patients with acute viral hepatitis, but that it is not detectable in patients with chronic HBV infection or uninfected healthy blood donors. Importantly, the peptide-activated CTL recognize target cells that express endogenously synthesized polymerase protein, suggesting that these peptides represent naturally processed viral epitopes. DNA sequence analysis of the viruses in patients who did not respond to peptide stimulation indicated that CTL nonresponsiveness was not due to infection by viral variants that differed in sequences from the synthetic peptides. CTL specific for one of the epitopes were unable to recognize several naturally occurring viral variants, except at high peptide concentration, underlining the HBV subtype specificity of this response. Furthermore, CTL responses against polymerase, core, and envelope epitopes were detectable for more than a year after complete clinical recovery and seroconversion, reflecting either the persistence of trace amounts of virus or the presence of long lived memory CTL in the absence of viral antigen. Finally, we demonstrated that wild type viral DNA and RNA can persist indefinitely, in trace quantities, in the serum and PBMC after complete clinical and serological recovery, despite a concomitant, vigorous, and sustained polyclonal CTL response. Since viral persistence is not due to escape from CTL recognition under these conditions, the data suggest that HBV may retreat into immunologically privileged sites from which it can seed the circulation and reach CTL-inaccessible tissues, thereby maintaining the CTL response in apparently cured individuals and, perhaps, prolonging the liver disease in patients with chronic hepatitis.

Acute Disease↗
PubMed1992

Design of polymerase chain reaction primers for the selective amplification of HIV-1 RNA in the presence of HIV-1 DNA.

OBJECTIVE: To develop a sensitive method for the specific detection of HIV-1 RNA. DESIGN: Following reverse transcription, the presence of HIV-1 RNA can be detected by polymerase chain reaction (PCR) amplification. Since specific detection of HIV-1 RNA may be complicated by contamination with minute quantities of HIV-1 DNA, samples are treated with deoxyribonuclease (DNase) prior to analysis. This additional step increases the possibility of RNA degradation and sample contamination. METHODS: A primer, HG141, was designed to hybridize to the poly(A) tract present in HIV-1 genomic and all HIV-1 messenger (m) RNA with its 5' end and to the region upstream of the poly(A) tract with its 3' end. The increased stability of the HG141 primer/HIV-1 RNA or complementary (c) DNA complex, enabled PCR amplification to be performed with HG141 and the return primer HG62 at an annealing temperature above the melting temperature (Tm) of the primer-HIV-1 DNA complex. RESULTS: After reverse transcription of samples obtained from HIV-1-infected H9 cells, HG62/141-primed PCR amplification specifically detected HIV-1 RNA sequences without the need for DNAase pre-treatment. This technique was more sensitive for the detection of HIV-1 RNA than SK38/39-primed PCR amplification of DNase-treated samples. CONCLUSIONS: Since the presence of HIV-1 RNA is indicative of HIV-1 replication for the presence of HIV-1 virions, the RNA-specific primer described should facilitate the assessment of HIV-1 replication and the plasma HIV-1 viral load in HIV-1-infected individuals. This should prove useful in the evaluation of the effects of therapeutic interventions on HIV-1 infection.

Base Sequence↗
PubMed2006

Assessment of the relationship between pre-chip and post-chip quality measures for Affymetrix GeneChip expression data.

BACKGROUND: Gene expression microarray experiments are expensive to conduct and guidelines for acceptable quality control at intermediate steps before and after the samples are hybridised to chips are vague. We conducted an experiment hybridising RNA from human brain to 117 U133A Affymetrix GeneChips and used these data to explore the relationship between 4 pre-chip variables and 22 post-chip outcomes and quality control measures. RESULTS: We found that the pre-chip variables were significantly correlated with each other but that this correlation was strongest between measures of RNA quality and cRNA yield. Post-mortem interval was negatively correlated with these variables. Four principal components, reflecting array outliers, array adjustment, hybridisation noise and RNA integrity, explain about 75% of the total post-chip measure variability. Two significant canonical correlations existed between the pre-chip and post-chip variables, derived from MAS 5.0, dChip and the Bioconductor packages affy and affyPLM. The strongest (CANCOR 0.838, p < 0.0001) correlated RNA integrity and yield with post chip quality control (QC) measures indexing 3'/5' RNA ratios, bias or scaling of the chip and scaling of the variability of the signal across the chip. Post-mortem interval was relatively unimportant. We also found that the RNA integrity number (RIN) could be moderately well predicted by post-chip measures B_ACTIN35, GAPDH35 and SF. CONCLUSION: We have found that the post-chip variables having the strongest association with quantities measurable before hybridisation are those reflecting RNA integrity. Other aspects of quality, such as noise measures (reflecting the execution of the assay) or measures reflecting data quality (outlier status and array adjustment variables) are not well predicted by the variables we were able to determine ahead of time. There could be other variables measurable pre-hybridisation which may be better associated with expression data quality measures. Uncovering such connections could create savings on costly microarray experiments by eliminating poor samples before hybridisation.

Artifacts↗
PubMed1997

Hygromycin B resistance mediates elimination of Leishmania virus from persistently infected parasites.

A series of pX63-HYG derivatives encoding Leishmania RNA virus 1-4 (LRV1-4) sequences were electroporated into cells of Leishmania strain M4147, a virus-infected strain of L. guyanensis. After 6 weeks of drug selection (hygromycin B), transfected parasites lacked detectable quantities of viral genomic double-stranded RNA, viral capsid protein, and RNA-dependent RNA polymerase (RDRP) activity. Evidence of viral infection was not recovered upon removal of the drug. While viral RNA transcripts were produced from electroporated expression vectors, as determined by reverse transcription-PCR, viral antigens were not detected, suggesting that the antiviral effects of hygromycin B are mediated through translation inhibition. A short-term selection study suggests that the LRV1-4 elimination may not only be a function of hygromycin B as a protein synthesis inhibitor but also possibly related to the mechanism of hygromycin B resistance in Leishmania strains.

Animals↗