scholarly journals Epigallocatechin Gallate Inhibits the HIV Reverse Transcription Step

2011 ◽  
Vol 21 (6) ◽  
pp. 239-243 ◽  
Author(s):  
Shenwei Li ◽  
Toshio Hattori ◽  
Eiichi N Kodama
Keyword(s):  
Reverse Transcription ◽  
Epigallocatechin Gallate ◽  
Reverse Transcription Step

scholarly journals PCR-based Calibration Curves for Studies of Quantitative Gene Expression in Human Monocytes: Development and Evaluation

2003 ◽  
Vol 49 (3) ◽  
pp. 425-432 ◽  
Author(s):  
Reidun Øvstebø ◽  
Kari Bente Foss Haug ◽  
Knut Lande ◽  
Peter Kierulf
Keyword(s):  
Total Variation ◽  
Reverse Transcription ◽  
Target Gene ◽  
Concentration Difference ◽  
Reverse Transcription Pcr ◽  
Calibration Curves ◽  
Reverse Transcription Step ◽  
Actin Mrna ◽  
Stringent Quality ◽  
Specific Mrna

Abstract Background: Quantitative reverse transcription-PCR (RT-PCR) used to detect small changes in specific mRNA concentrations is often associated with poor reproducibility. Thus, there is a need for stringent quality control in each step of the protocol. Methods: Real-time PCR-based calibration curves for a target gene, tissue factor (TF), and a reference gene, β-actin, generated from PCR amplicons were evaluated by running cDNA controls. In addition, the reverse transcription step was evaluated by running mRNA controls. Amplification efficiencies of calibrators and targets were determined. Variances within and between runs were estimated, and power statistics were applied to determine the concentration differences that could reliably be detected. Results: Within- and between-run variations (CVs) of cDNA controls (TF and β-actin), extrapolated from reproducible calibration curves (CVs of slopes, 4.3% and 2.7%, respectively) were 4–10% (within) and 15–38% (between) using both daily and “grand mean” calibration curves. CVs for the β-actin mRNA controls were 12% (within) and 19–28% (between). Estimates of each step’s contribution to the total variation were as follows: CVRT-PCR, 28%; CVPCR, 15%; CVRT, 23% (difference between CVRT-PCR and CVPCR). PCR efficiencies were as follows: β-actin calibrator/target, 1.96/1.95; TF calibrator/target, 1.95/1.93. Duplicate measurements could detect a twofold concentration difference (power, 0.8). Conclusions: Daily PCR calibration curves generated from PCR amplicons were reproducible, allowing the use of a grand mean calibration curve. The reverse transcription step contributes the most to the total variation. By determining a system’s total variance, power analysis may be used to disclose differences that can be reliably detected at a specified power.

scholarly journals USP18 (UBP43) Abrogates p21-Mediated Inhibition of HIV-1

2018 ◽  
Vol 92 (20) ◽  
Author(s):  
Edmund Osei Kuffour ◽  
Kerstin Schott ◽  
Ananda Ayyappan Jaguva Vasudevan ◽  
Jessica Holler ◽  
Wolfgang A. Schulz ◽  
...  
Keyword(s):  
Protein Expression ◽  
Reverse Transcription ◽  
Kinase Inhibitor ◽  
Active Form ◽  
Innate Immune ◽  
P21 Protein ◽  
Intracellular Dntp ◽  
Antiviral Function ◽  
Reverse Transcription Step ◽  
Hiv 1

ABSTRACTThe host intrinsic innate immune system drives antiviral defenses and viral restriction, which includes the production of soluble factors, such as type I and III interferon (IFN), and activation of restriction factors, including SAMHD1, a deoxynucleoside triphosphohydrolase. Interferon-stimulated gene 15 (ISG15)-specific ubiquitin-like protease 43 (USP18) abrogates IFN signaling pathways. The cyclin-dependent kinase inhibitor p21 (CIP1/WAF1), which is involved in the differentiation and maturation of monocytes, inhibits human immunodeficiency virus type 1 (HIV-1) in macrophages and dendritic cells. p21 inhibition of HIV-1 replication is thought to occur at the reverse transcription step, likely by suppressing cellular deoxynucleoside triphosphate (dNTP) biosynthesis and increasing the amount of antivirally active form of SAMHD1. SAMHD1 strongly inhibits HIV-1 replication in myeloid and resting CD4+T cells. Here, we studied how USP18 influences HIV-1 replication in human myeloid THP-1 cells. We found that USP18 has the novel ability to inhibit the antiviral function of p21 in differentiated THP-1 cells. USP18 enhanced reverse transcription of HIV-1 by downregulating p21 expression and upregulating intracellular dNTP levels. p21 downregulation by USP18 was associated with the active form of SAMHD1, phosphorylated at T592. USP18 formed a complex with the E3 ubiquitin ligase recognition factor SKP2 (S-phase kinase associated protein 2) and SAMHD1. CRISPR-Cas9 knockout of USP18 increased p21 protein expression and blocked HIV-1 replication. Overall, we propose USP18 as a regulator of p21 antiviral function in differentiated myeloid THP-1 cells.IMPORTANCEMacrophages and dendritic cells are usually the first point of contact with pathogens, including lentiviruses. Host restriction factors, including SAMHD1, mediate the innate immune response against these viruses. However, HIV-1 has evolved to circumvent the innate immune response and establishes disseminated infection. The cyclin-dependent kinase inhibitor p21, which is involved in differentiation and maturation of monocytes, blocks HIV-1 replication at the reverse transcription step. p21 is thought to suppress key enzymes involved in dNTP biosynthesis and activates SAMHD1 antiviral function. We report here that the human USP18 protein is a novel factor potentially contributing to HIV replication by blocking the antiviral function of p21 in differentiated human myeloid cells. USP18 downregulates p21 protein expression, which correlates with upregulated intracellular dNTP levels and the antiviral inactive form of SAMHD1. Depletion of USP18 stabilizes p21 protein expression, which correlates with dephosphorylated SAMHD1 and a block to HIV-1 replication.

scholarly journals Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

2020 ◽  
Author(s):  
Fabien Cholet ◽  
Umer Z. Ijaz ◽  
Cindy J. Smith
Keyword(s):  
16S Rrna ◽  
Reverse Transcription ◽  
Environmental Microbiology ◽  
High Sensitivity ◽  
Amplicon Sequencing ◽  
Rt Pcr ◽  
Reverse Transcriptases ◽  
Reverse Transcriptase Enzyme ◽  
Reverse Transcription Step ◽  
Gene Expression Studies

SummaryRT-Q-PCR, and RT-PCR amplicon sequencing, provide a convenient, target-specific, high-sensitivity approach for gene expression studies and are widely used in environmental microbiology. Yet, the effectiveness and reproducibility of the reverse transcription step has not been evaluated. Therefore, we tested a combination of four commercial reverse transcriptases with two priming techniques to faithfully transcribe 16S rRNA and amoA transcripts from marine sediments. Both enzyme and priming strategy greatly affected quantification of the exact same target with differences of up to 600-fold. Furthermore, the choice of RT system significantly changed the communities recovered. For 16S rRNA, both enzyme and priming had a significant effect with enzyme having a stronger impact than priming. Inversely, for amoA only the change in priming strategy resulted in significant differences between the same sample. Specifically, more OTUs and better coverage of amoA transcripts diversity were obtained with GS priming indicating this approach was better at recovering the diversity of amoA transcripts. Moreover, sequencing of RNA mock communities revealed that, even though transcript α diversities (i.e. OTU counts within a sample) can be biased by the RT, the comparison of β diversities (i.e. differences in OTU counts between samples) is reliable as those biases are reproducible between environments.Originality-Significance StatementIs the complementary DNA (cDNA) produced after Reverse Transcription (RT) a faithful representation of the starting RNA? This is a fundamental and important question for transcriptomic-based studies in environmental microbiology that aim to quantify and/or examine the diversity of transcripts via RT approaches. Yet little is known about the reliability and reproducibility of this step. Here, we evaluated the effect of the two main components of the RT reaction – the retro transcriptase enzyme and priming strategy (gene specific vs random priming), on the quantification and diversity of cDNA. We found that both have a significant impact. We further provide evidence to enable informed choices as to the enzyme and priming combinations to improve the performance of RT-PCR approaches. Taken together, this work will improve the reliability and reproducibility of transcript-based studies in environmental microbiology.

scholarly journals DNA display of folded RNA libraries enabling RNA-SELEX without reverse transcription

2017 ◽  
Vol 53 (19) ◽  
pp. 2878-2881 ◽  
Author(s):  
I. S. MacPherson ◽  
J. S. Temme ◽  
I. J. Krauss
Keyword(s):  
Reverse Transcription ◽  
Reverse Transcription Step ◽  
Systematic Evolution ◽  
Rna Ligands ◽  
Exponential Enrichment ◽  
Folded Rna

A method for the physical attachment of folded RNA libraries to their encoding DNA is presented as a way to circumvent the reverse transcription step during systematic evolution of RNA ligands by exponential enrichment (RNA-SELEX).

scholarly journals Lv2, a Novel Postentry Restriction, Is Mediated by both Capsid and Envelope

2004 ◽  
Vol 78 (4) ◽  
pp. 2006-2016 ◽  
Author(s):  
Christian Schmitz ◽  
David Marchant ◽  
Stuart J. D. Neil ◽  
Keith Aubin ◽  
Sandra Reuter ◽  
...  
Keyword(s):  
Life Cycle ◽  
Reverse Transcription ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Nuclear Entry ◽  
Cellular Compartment ◽  
Glycoprotein G ◽  
Reverse Transcription Step ◽  
Viral Determinants ◽  
Hiv 1

ABSTRACT The characterization of restrictions to lentivirus replication in cells identifies critical steps in the viral life cycle and potential therapeutic targets. We previously reported that a human immunodeficiency virus type 2 (HIV-2) isolate was restricted to infection in some human cells, which led us to identify a step in the life cycle of HIV-2 detected after reverse transcription but prior to nuclear entry. The block is bypassed with a vesicular stomatitis virus glycoprotein G (VSV-G) envelope (A. McKnight et al., J. Virol. 75:6914-6922, 2001). We hypothesized that, although the restriction is apparent at a post-reverse transcription step, the lack of progress results from a failure of the virus to reach a cellular compartment with access to the nucleus. Here we analyzed molecular clones of the restricted virus, MCR, and an unrestricted virus, MCN. Using sequence analysis and gene swapping, we mapped the viral determinants to gag and env. Site-directed mutagenesis identified a single amino acid at position 207 in CA to be responsible for the gag restriction. Pseudotype experiments indicate that this step is also important for the infection of cells by HIV-1. The HIV-1 NL4.3 core is restricted if supplied with a restricted MCR envelope but not with VSV-G. Also the NL4.3 envelope rescues the restricted core of HIV-2 MCR. Abrogation experiments with MLV demonstrate that the restriction is distinct from Fv1/Ref1/Lv1. We propose that this represents a new lentiviral restriction, Lv2. Thus, the envelope and capsid of HIV act to ensure that the virus is delivered into an appropriate cellular compartment that allows postentry events in viral replication to proceed efficiently.

scholarly journals Improvement in the sensitivity of viroid detection by adapting the reverse transcription step in one-step RT-qPCR assays

2021 ◽  
Vol 292 ◽  
pp. 114123
Author(s):  
Thomas Leichtfried ◽  
Helga Reisenzein ◽  
Siegrid Steinkellner ◽  
Richard A. Gottsberger
Keyword(s):  
Reverse Transcription ◽  
Reverse Transcription Step ◽  
One Step

scholarly journals Properties of the Reverse Transcription Reaction in mRNA Quantification

2004 ◽  
Vol 50 (3) ◽  
pp. 509-515 ◽  
Author(s):  
Anders Ståhlberg ◽  
Joakim Håkansson ◽  
Xiaojie Xian ◽  
Henrik Semb ◽  
Mikael Kubista
Keyword(s):  
Gene Expression ◽  
Reverse Transcription ◽  
Experimental Accuracy ◽  
Reaction Conditions ◽  
Quantitative Measurements ◽  
Experimental Variation ◽  
Reverse Transcription Reaction ◽  
Transcription Efficiency ◽  
Reverse Transcription Step ◽  
Rna Concentration

Abstract Background: In most measurements of gene expression, mRNA is first reverse-transcribed into cDNA. We studied the reverse transcription reaction and its consequences for quantitative measurements of gene expression. Methods: We used SYBR green I-based quantitative real-time PCR (QPCR) to measure the properties of reverse transcription reaction for the β-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and insulin II genes, using random hexamers, oligo(dT), and gene-specific reverse transcription primers. Results: Experimental variation in reverse transcription-QPCR (RT-QPCR) was mainly attributable to the reverse transcription step. Reverse transcription efficiency depended on priming strategy, and the dependence was different for the five genes studied. Reverse transcription yields also depended on total RNA concentration. Conclusions: RT-QPCR gene expression measurements are comparable only when the same priming strategy and reaction conditions are used in all experiments and the samples contain the same total amount of RNA. Experimental accuracy is improved by running samples in (at least) duplicate starting with the reverse transcription reaction.

scholarly journals Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription: a ‘zero-step’ RT-qPCR protocol

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Petra Chovancova ◽  
Verena Merk ◽  
Andreas Marx ◽  
Marcel Leist ◽  
Ramon Kranaster
Keyword(s):  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Cultured Cells ◽  
Rna Extraction ◽  
Gene Target ◽  
Human Neurons ◽  
Reverse Transcription Quantitative Pcr ◽  
Reverse Transcription Step ◽  
Short Time ◽  
Quantify Gene Expression

Abstract We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and without an isothermal reverse-transcription step. Human neurons (Lund human mesencephalic cells) were lysed at different stages of differentiation, and the lysates were used directly as template for the combined RT-qPCR reaction. We detected a down-regulation of a proliferation marker and an up-regulation of neuronal dopaminergic genes expression. We were able to detect the reference gene target from as few as a single cell, demonstrating the application of the method for efficient amplification from small cell numbers. The data were fully in line with those obtained by the standard two-step RT-qPCR from the extracted total RNA. Our ‘zero-step’ RT-qPCR method proved to be simple and reliable with a total time from cell lysis to the end of the qPCR as short as 1.5 h. It is therefore particularly suitable for RT-qPCRs where large numbers of samples must be handled, or where data are required within short time.

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