Reverse Transcription Step Research Articles

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS)

A one step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of viral hemorrhagic septicaemia virus (VHS). A set of six primers were designed, based on the G-protein sequence of the VHS virus serotypes (He, F1, 23.75, Klapmolle and Rindsholm). The assay was optimised to amplify VHS RNA by incubation at 63 °C for only 1 h, and required only a simple water bath or heating block to provide a constant temperature of 63 °C. RT-LAMP amplification products were detected by visual inspection using SYBR Green I stain and had a ladder-like appearance when electrophoresed on an agarose gel. The detection limit of the RT-LAMP assay was found to be similar to the commonly used RT-PCR method: both methods detected VHS RNA at a dilution of 10 6. The assay was evaluated using clinical samples and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for VHS virus.

Single-Molecule FRET Studies of Important Intermediates in the Nucleocapsid-Protein-Chaperoned Minus-Strand Transfer Step in HIV-1 Reverse Transcription

The minus-strand transfer step of HIV-1 reverse transcription is chaperoned by the nucleocapsid protein (NC), which has been shown to facilitate the annealing between the transactivation response element (TAR) RNA and complementary TAR DNA stem-loop structures. In this work, potential intermediates in the mechanism of NC-chaperoned TAR DNA/TAR RNA annealing have been examined using single-molecule fluorescence resonance energy transfer. The interaction between TAR DNA and various DNA oligonucleotides designed to mimic the initial annealing step was monitored to capture potential intermediates along the reaction pathway. Two possible mechanisms of annealing were examined, namely nucleation through the 3′/5′ termini, termed the “zipper” complex, or nucleation through the hairpin loops in a “kissing” complex. Intermediates associated with both mechanisms were observed in the presence of NC, and the kinetics of formation of these intermediates were also measured. Thus, the single-molecule experiments support the notion that NC-assisted annealing of TAR DNA:TAR RNA may occur through multiple pathways.

Comment on Wong and Medrano’s “Real-Time PCR for mRNA Quantification” BioTechniques 39 :75-85 (July 2005)

quantitative PCR was introduced in the beginning of the 1990s, several groups used internal synthetic standards in the reactions to account for variances in reaction efficiencies, but these are not mentioned, suggesting that they have fallen out of fashion. These standards were typically identical to the target but lacked a sequence in the middle to be able to distinguish the standard from the target. Most importantly, they had the same primer sequences as the target, indicating simple multiplexing of the standard and the target. These internal standards can be used to absolutely quantify mRNA targets (2) and could solve many of the problems identified in the article by Wong and Medrano. It is easy to imagine such a standard in real-time PCR where the two PCR products (standard and target) could be separated by probes emitting different colors. Synthetic RNA internal standards have three advantages. First, by spiking the RNA sample with known amounts of standards, efficiencies in the reverse transcription step is accounted for. Since the standard is converted into cDNA, differences between tubes in the efficiency of the PCR are accounted for as well. Second, there is no need to worry about which reference gene to use for normalization. Third, absolute quantification could easily be obtained by relating to a known amount of internal standard (2) instead of relating to a relative internal standard, as in the case of a housekeeping gene and an external standard curve. It is not difficult to understand why such synthetic RNA standards are not used today since it takes some effort to produce and handle them without degradation, but it is difficult to understand why they are not considered as a solution or a tool for mRNA quantification using real-time PCR.

Mechanisms of HIV-1 Inhibition by the Lipid Mediator N-Arachidonoyldopamine

Several linear fatty acid dopamides (N-acyldopamines) have been identified recently in the brain. Among them, N-arachidonoyldopamine (NADA) is an endogenous lipid mediator sharing endocannabinoid and endovanilloid biological activities. We have reported previously that NADA exerts some of its biological activities through inhibition of the NF-kappaB pathway and, because this transcription factor plays a key role in HIV-1-long terminal repeat (LTR) trans activation, we have evaluated the anti-HIV-1 activity of NADA. In this study, we show that NADA inhibits vesicular stomatitis virus-pseudotyped HIV-1 infection in the human leukemia T cell line Jurkat, in primary T cells, and in the human astrocytic cell line U373-MG. Other endocannabinoids such as anandamide, 2-arachidonoylglycerol, and noladin ether did not show inhibitory activity in the HIV-1 replication assays. The anti-HIV-1 activity of NADA was independent of known cannabinoid and vanilloid receptor activation. In addition, NADA did not affect reverse transcription and integration steps of the viral cycle, and its inhibitory effect was additive with that of the reverse transcriptase inhibitor azidothymidine. NADA inhibited both TNF-alpha and HIV-1 trans activator protein-induced HIV-1-LTR activation. We also show that NADA counteracts the TNF-alpha-mediated trans activation capacity of the p65 NF-kappaB subunit without affecting its physical association to the HIV-1-LTR promoter. Moreover, NADA inhibited the p65 transcriptional activity by specifically targeting the phosphorylation of this NF-kappaB subunit at Ser(536). These findings provide new mechanistic insights into the biological activities of NADA, and highlight the potential of lipid mediators for the management of AIDS.

A Synthetic Glycosaminoglycan Mimetic Binds Vascular Endothelial Growth Factor and Modulates Angiogenesis

In a previous study, we showed that in situ injection of glycosaminoglycan mimetics called RGTAs (ReGeneraTing Agents) enhanced neovascularization after skeletal muscular ischemia (Desgranges, P., Barbaud, C., Caruelle, J. P., Barritault, D., and Gautron, J. (1999) FASEB J. 13, 761-766). In the present study, we showed that the RGTA OTR4120 modulated angiogenesis in the chicken embryo chorioallantoic membrane assay, in a dose-dependent manner. We therefore investigated the effect of OTR4120 on one of the most specific angiogenesis-regulating heparin-binding growth factors, vascular endothelial growth factor 165 (VEGF165). OTR4120 showed high affinity binding to VEGF165 (Kd = 2.2 nm), as compared with heparin (Kd = 15 nm), and potentiated the affinity of VEGF165 for VEGF receptor-1 and -2 and for neuropilin-1. In vitro, OTR4120 potentiated VEGF165-induced proliferation and migration of human umbilical vein endothelial cells. In the in vivo Matrigel plug angiogenesis assay, OTR4120 in a concentration as low as 3 ng/ml caused a 6-fold increase in VEGF165-induced angiogenesis. Immunohistochemical staining showed a larger number of well differentiated VEGFR-2-expressing-cells in Matrigel sections of OTR4120-treated plug than in control sections. These findings indicate that OTR4120 enhances the VEGF165-induced angiogenesis and therefore may hold promise for treating disorders characterized by deficient angiogenesis.

Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays

Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at -20 degrees C, 4 degrees C, and room temperature was demonstrated.

A novel frameshift mutation (+G) at codons 15/16 in a β0 thalassaemia gene results in a significant reduction of β globin mRNA values

Aims: To identify a novel β globin gene mutation found in a Chinese family, and also to assess its functional consequences. Methods: Haematological analysis was performed on all family members....

Retroviral superinfection resistance

The retroviral phenomenon of superinfection resistance (SIR) defines an interference mechanism that is established after primary infection, preventing the infected cell from being superinfected by a similar type of virus. This review describes our present understanding of the underlying mechanisms of SIR established by three characteristic retroviruses: Murine Leukaemia Virus (MuLV), Foamy Virus (FV), and Human Immunodeficiency Virus (HIV). In addition, SIR is discussed with respect to HIV superinfection of humans.MuLV resistant mice exhibit two genetic resistance traits related to SIR. The cellular Fv4 gene expresses an Env related protein that establishes resistance against MuLV infection. Another mouse gene (Fv1) mediates MuLV resistance by expression of a sequence that is distantly related to Gag and that blocks the viral infection after the reverse transcription step. FVs induce two distinct mechanisms of superinfection resistance. First, expression of the Env protein results in SIR, probably by occupancy of the cellular receptors for FV entry. Second, an increase in the concentration of the viral Bet (Between-env-and-LTR-1-and-2) protein reduces proviral FV gene expression by inhibition of the transcriptional activator protein Tas (Transactivator of spumaviruses). In contrast to SIR in FV and MuLV infection, the underlying mechanism of SIR in HIV-infected cells is poorly understood. CD4 receptor down-modulation, a major characteristic of HIV-infected cells, has been proposed to be the main mechanism of SIR against HIV, but data have been contradictory. Several recent studies report the occurrence of HIV superinfection in humans; an event associated with the generation of recombinant HIV strains and possibly with increased disease progression. The role of SIR in protecting patients from HIV superinfection has not been studied so far.The phenomenon of SIR may also be important in the protection of primates that are vaccinated with live attenuated simian immunodeficiency virus (SIV) against pathogenic SIV variants. As primate models of SIV infection closely resemble HIV infection, a better knowledge of SIR-induced mechanisms could contribute to the development of an HIV vaccine or other antiviral strategies.

Straightforward Procedure for Internal Control of Real-Time Reverse Transcription Amplification Assays

Internal controls are crucial for reliable detection of pathogens in nucleic acid amplification assays. Without an internal control, a negative amplification result may mean that the sample contains no target or that the reaction failed. We recently described (1) the use of internal controls that consist of internal control oligonucleotides (ICOs) that contain little more than primer sites and a probe-binding site that contains mismatches to the actual target. ICOs can be obtained commercially and do not require a separate control-specific detection probe. Because of the mismatches, the control does not hybridize to the single detection probe during the fluorescence-acquisition step, and thus the probe binds and detects only nucleic acid derived from the pathogen. However, amplification of the ICO can be verified during melting analysis after PCR. Many important viral pathogens contain an RNA genome that must be transcribed to cDNA before amplification. To provide a control for both reverse transcription and amplification, an internal control must be an RNA molecule. The standard method for generating such a control involves directional cloning of a double-stranded DNA template into a plasmid vector carrying bacteriophage RNA polymerase promoter sequences. The RNA is then transcribed in vitro from the DNA template (2)(3). We have extended the ICO technique to RNA by introducing an RNA polymerase promoter and adding 2 simple preparation steps. We demonstrate the application of this control technique to the reverse transcription-PCR (RT-PCR) step of a LightCycler (Roche Molecular Biochemicals) real-time hybridization probe assay for respiratory syncytial virus (RSV). The sequences of the primers and probes for the RSV assay were 5′-GCCAAAAAATTGTTTCCACAATA-3′, 5′-TCTTCATCACCATACTTTTCTGTTA-3′, 5′-CY5.5-GGAATTCACATGGTCTACTACTGACTGT-phosphate, and 5′-GTTGTTCTATAAGCTGGTATTGATGCA-fluorescein-3′ (4). The 2-step LightCycler (Roche) RT-PCR was performed as follows. For the reverse transcription step (total volume of 10 μL), the reaction mixture consisted of 1.5 mM MgCl2, 2.5 …

Evaluation of an Affymetrix High‐density Oligonucleotide Microarray Platform as a Measurement System

AbstractThe random measurement or technological variation of an Affymetrix high‐density oligonucleotide microarray platform is estimated and evaluated against the variation in the gene expression levels of a biological sample. A mixed effects analysis of variance model is used to describe the probe intensity levels. The focus is on the repeatability of the method. Additionally, the minimal number of arrays necessary to detect biological relevant differences in gene expression levels between biological samples is estimated based on the repeatability. The investigated sources of variation for the repeatability are arrays, array production lots, the reverse transcription step, the in vitro transcription step and the hybridization and washing step. They may all add to the short‐term random measurement variation. The effect of global linear normalization on the repeatability is determined. It is shown that the in vitro transcription step contributes to the repeatability with more than 50%. If normalization is used, the microarray platform is indeed suitable for measuring thousands of genes simultaneously and in particular to detect differences in gene expression between biological samples. Based on a goodness‐of‐fit investigation and considering the amount of data involved, the analysis of variance model seems suitable to describe the raw probe intensity data. Copyright © 2005 John Wiley & Sons, Ltd.

Simple Enzymatic Means to Neutralize DNA Contamination in Nucleic Acid Amplification

Reverse transcription PCR (RT-PCR) is prone to false positives when contaminating DNA molecules are present at the start of a reaction. Contaminants that derive from earlier work using a given primer pair (carryover PCR products) are of particular concern when those primers are used routinely, as in clinical diagnostics or environmental monitoring. In addition, contamination by genomic DNA can significantly interfere with quantitative and qualitative analysis of RNAs by RT-PCR. Here we describe contaminant restriction (ConR), a method that can be used to neutralize carryover and genomic DNA contamination in RT-PCR studies. Restriction enzymes (REs) added to the amplification cocktail cleave contaminant DNA molecules while sparing the intended target nucleic acid. Restriction, reverse transcription, and amplification steps all take place in the same sealed vessel, thus avoiding any danger of recontamination. ConR eliminates carryover contamination in PCR without compromising target sequence amplification. Because the method is effective against both genomic and carryover contamination, it can be employed routinely in one-step RT-PCR, whatever the RNA target or the nature of the potential DNA contaminant. A variation of this decontamination method, amplicon primer site restriction (APSR), is effective specifically against carryover contamination. APSR, unlike ConR, can be applied during PCR-based amplification of DNA target molecules.

An Internal Reference Technique for Accurately Quantifying Specific mRNAs by Real-Time PCR with Application to the tceA Reductive Dehalogenase Gene

The accuracy of mRNA quantification by reverse transcription (RT) in conjunction with real-time PCR (qPCR) is limited by mRNA losses during sample preparation (cell lysis, RNA isolation, and DNA removal) and by inefficiencies in reverse transcription. To control for these losses and inefficiencies, a technique was developed that utilizes an exogenous internal reference mRNA (ref mRNA) along with mRNA absolute standard curves. The technique was applied to quantify mRNA of the trichloroethene (TCE) reductive dehalogenase-encoding tceA gene in an anaerobic TCE-to-ethene dechlorinating microbial enrichment. Compared to RT-qPCR protocols that utilize DNA absolute standard curves, application of the new technique increased measured quantities of tceA mRNA by threefold, demonstrating a substantial improvement in quantification. The technique was also effective for quantifying the loss of mRNA during specific steps of the sample processing protocol. Analysis revealed that the efficiency of the RNA isolation (56%) step was significantly less than that of the cell lysis (84%), DNA removal (93%), and RT (88%) steps. The technique was applied to compare the effects of cellular exposure to different chlorinated ethenes on tceA expression. Results show that exposure to TCE or cis-1,2-dichloroethene resulted in 25-fold-higher quantities of tceA mRNA than exposure to vinyl chloride or chlorinated ethene starvation.

Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo

Because the replication of hepatitis B virus (HBV) proceeds via an obligatory reverse transcription step in the viral capsid, cDNA is potentially vulnerable to editing by cytidine deaminases of the APOBEC3 family. To date only two edited HBV genomes, referred to as G --> A hypermutants, have been described in vivo. Recent work suggested that HBV replication was indeed restricted by APOBEC3G but by a mechanism other than editing. The issue of restriction has been explored by using a sensitive PCR method allowing differential amplification of AT-rich DNA. G --> A hypermutated HBV genomes were recovered from transfection experiments involving APOBEC3B, -3C, -3F, and -3G indicating that all four enzymes were able to extensively deaminate cytidine residues in minus-strand DNA. Unexpectedly, three of the four enzymes (APOBEC3B, -3F, and -3G) deaminated HBV plus-strand DNA as well. From the serum of two of four patients with high viremia, G --> A hypermutated genomes were recovered at a frequency of approximately 10(-4), indicating that they are, albeit relatively rare, part of the natural cycle of HBV infection. These findings suggest that human APOBEC3 enzymes can impact HBV replication via cytidine deamination.

Mesuol, a natural occurring 4-phenylcoumarin, inhibits HIV-1 replication by targeting the NF-κB pathway

Coumarins and structurally related compounds have been recently shown to inhibit replication of human immunodeficiency virus (HIV) and thus, exhibit a therapeutic potential. In this study we report that mesuol and isomesuol, two 4-phenyl coumarins, isolated from the tree Marila pluricostata, suppress HIV-1 replication in Jurkat T cells. These coumarins do not affect the reverse transcription and intregration steps of the viral cycle and their antiviral effect is additive with that of azidothymidine (AZT). In addition, mesuol inhibits TNFα-induced HIV-1-LTR transcriptional activity by targeting the nuclear factor-κB (NF-κB) pathway. While mesuol does not prevent either the binding of NF-κB to DNA or the phosphorylation and degradation of NF-κB inhibitory protein, IκBα, it inhibits the phosphorylation and the transcriptional activity of the NF-κB p65 subunit in TNFα-stimulated cells. These results highlight the potential of the NF-κB transcription factor as a target for anti-HIV-1 compounds such as 4-phenyl coumarins, which could serve as lead compounds for the development of additional therapeutic approaches against AIDS.

Translation Toeprinting Assays Using Fluorescently Labeled Primers and Capillary Electrophoresis

The protocol described here is an adapted version of the toeprinting assay in which the oligonucleotide used to prime the reverse transcription step is labeled with a fluorescent dye instead of 32P. By using a fluorescent dye, the results of the assay are obtained within one hour by direct electrophoresis of the samples on an automated sequencing machine. This eliminates the need to cast and run polyacrylamide gels and to wait to expose dried gels. We show that an identical toeprint was found for the chloramphenicol acetyltransferase transcript using this nonradioactive method, which is in agreement with the previously published 32P-labeled method. Furthermore, in addition to being a faster and safer method, a larger region of sequence can be analyzed with one primer in a single experiment.

Inhibition of Human Immunodeficiency Virus Type-1 (HIV-1) Replication at a Reverse Transcription Step by Human Cell Factor(s)

Infection of human cell with human immunodeficiency virus type-1 (HIV-1) was suppressed by cellular genetic factor(s) at reverse transcription step. Although same amount of virus adsorbed on both cells, small amount of HIV-1 (IIIB strain) infected HeLa (MAGI/CCR5) cell, while large amount of HIV-1 infected HOS (GHOST/CXCR4) cell. Regulation of virus replication at postentry level by cellular factor(s) had an important role for low efficiency of HIV-1 infection to MAGI/CCR5 cell. Provirus DNA formation in MAGI/CCR5 cell was less efficient than in GHOST/CXCR4 cell. Once GHOST/CXCR4 cell was fused with MAGI/CCR5 cell, susceptibility against HIV-1 decreased. Further, HIV-1 reverse transcriptase (RT) activity was strongly inhibited by cytosolic protein, derived from MAGI/CCR5 cell, in vitro. This research cleared a certain human cell genetically carries some factor(s) which inhibits the activity of HIV-1 RT.

Hepatitis B Virus e Antigen Variants

More than 300 million people worldwide are chronically infected with hepatitis B virus (HBV). Considering the very short generation time for a virus, and the high error rate associated with the reverse transcription step of HBV replication, decades of HBV infection are probably equivalent to million years of human evolution. The most important selective force during the natural course of HBV infection appears to be the immune response. The development of anti-HBe antibody in hepatitis B patients usually correlates with reduction of HBV viremia. As a consequence, escape mutants of anti-HBe are selected. The core promoter mutants express less HBe antigen (HBeAg) through transcriptional down regulation, while precore mutants express truncated products. We recently identified additional mutations that modulate HBeAg translation initiation, proteolytic cleavage, and secondary structure maintenance through a disulfide bond. The core promoter mutants have been associated with the development of fulminant hepatitis during acute infection and liver cancer during chronic infection. Consistent with their enhanced pathogenicity, core promoter mutants were found to replicate at up to 10-fold higher levels in transfected human hepatoma cells than the wild-type virus. Moreover, some core promoter mutants are impaired in virion secretion due to missense mutations in the envelope gene. These virological properties may help explain enhanced pathogenicity of core promoter mutants in vivo.

Development of a TaqMan ® RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses

Chikungunya virus (CHIKV), a member of the alphavirus genus, is of considerable public health concern in Southeast Asian and African countries. However, despite serological evidence, the diagnosis of this arthropod-borne human disease is confirmed infrequently and needs to be improved. In fact, illness caused by CHIKV can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms, and laboratory confirmation of suspected cases is required to launch control measures during an epidemic. Moreover, no quantitative molecular tool is described to study CHIKV replication or detection in clinical samples and cell culture supernatants. In this study, a specific and sensitive CHIKV one-step TaqMan ® RT-PCR assay was developed as a tool for the diagnosis of African CHIKV as well as a rapid indicator of active infection by quantifying viral load. This study also showed that a simple heat viral RNA release during the reverse transcription step constituted an alternative to the conventional RNA extraction method.

Direct Measurement of Multiple mRNAs in Nerve Growth Factor-Induced PC12 Cells Using Electrophoretic Tags to Monitor Biomarkers of Neuronal Differentiation in 96-Well Format

Pheochromocytoma-12 (PC12) cells recapitulate the program of neuronal differentiation by developing neurites after about 12 days of nerve growth factor (NGF) treatment. This model can be used to evaluate the neuroprotective/neurotrophic effect of compounds. Specific mRNAs such as cfos and c-jun are early biomarkers of the irreversible commitment into the differentiation program as they appear after only 30-40 min of NGF treatment. Monitoring the level of these mRNAs instead of the neurite outgrowth dramatically reduces the time needed to identify the drug potential of compounds. The electrophoretic tags, or eTag reporters (ACLARA Biosciences, Inc., Mountain View, CA), are a new class of fluorescent reporters that have unique migration properties in capillary electrophoresis, which allows for their separation and identification. (The eTag Multiplex Invader Assay and products incorporate Invader technology and Cleavase enzyme licensed for use from Third Wave Technologies, Inc. [Madison, WI] for multiplexed gene expression applications.) Each eTag molecule used begins as a phosphoramidite that is incorporated into a specific oligonucleotide using standard oligonucleotide synthesis procedures. A set of distinct probes labeled with different eTag molecules can then be mixed together to simultaneously quantify the levels of different mRNAs from the same sample. When compared to existing methods for measuring multiplexed gene expression from the same sample, the eTag assay allows a direct quantification of the mRNA from cells without any extraction/purification and still provides multiplexing capability, high sensitivity, miniaturization, and reproducibility compatible with medium-throughput screening methods. The eTag technology was used to simultaneously measure the level of expression of four mRNAs-c-fos, c-jun, c-myc, and gapdh-in NGF-treated PC12 cells in a standard 96-well format. The experimental data shown here demonstrate the use of eTag technology as a new screening tool, which uniquely combines robustness, sensitivity, multiplexing capability, and direct measurement of mRNA without any sample preparation steps, such as RNA extraction/purification or a reverse transcription step.

Human APOBEC3F is another host factor that blocks human immunodeficiency virus type 1 replication.

Recently, APOBEC3G has been identified as a host factor that blocks retroviral replication. It introduces G to A hypermutations in newly synthesized minus strand viral cDNA at the step of reverse transcription in target cells. Here, we identified the human APOBEC3F protein as another host factor that blocks human immunodeficiency virus type 1 (HIV-1) replication. Similar to APOBEC3G, APOBEC3F also induced G to A hypermutations in HIV genomic DNA, and the viral Vif protein counteracted its activity. Thus, APOBEC family members might have evolved as a general defense mechanism of the body against retroviruses, retrotransposons, and other mobile genetic elements.