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Cells Sep 2021Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity,... (Review)
Review
Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.
Topics: Gene Expression Profiling; Humans; Real-Time Polymerase Chain Reaction; Reverse Transcription; Sensitivity and Specificity; Single-Cell Analysis
PubMed: 34685587
DOI: 10.3390/cells10102607 -
Science (New York, N.Y.) Apr 2023Non-long terminal repeat (non-LTR) retrotransposons, or long interspersed nuclear elements (LINEs), are an abundant class of eukaryotic transposons that insert into...
Non-long terminal repeat (non-LTR) retrotransposons, or long interspersed nuclear elements (LINEs), are an abundant class of eukaryotic transposons that insert into genomes by target-primed reverse transcription (TPRT). During TPRT, a target DNA sequence is nicked and primes reverse transcription of the retrotransposon RNA. Here, we report the cryo-electron microscopy structure of the R2 non-LTR retrotransposon initiating TPRT at its ribosomal DNA target. The target DNA sequence is unwound at the insertion site and recognized by an upstream motif. An extension of the reverse transcriptase (RT) domain recognizes the retrotransposon RNA and guides the 3' end into the RT active site to template reverse transcription. We used Cas9 to retarget R2 in vitro to non-native sequences, suggesting future use as a reprogrammable RNA-based gene-insertion tool.
Topics: Cryoelectron Microscopy; Long Interspersed Nucleotide Elements; Reverse Transcription; RNA-Directed DNA Polymerase; Bombyx
PubMed: 37023171
DOI: 10.1126/science.adg7883 -
Current Opinion in Structural Biology Apr 2015Antiviral restriction factors are an integral part of the host innate immune system that protects cells from viral pathogens, such as human immunodeficiency virus (HIV).... (Review)
Review
Antiviral restriction factors are an integral part of the host innate immune system that protects cells from viral pathogens, such as human immunodeficiency virus (HIV). Studies of the interactions between restriction factors and HIV have greatly advanced our understanding of both the viral life cycle and basic cell biology, as well as provided new opportunities for therapeutic intervention of viral infection. Here we review the recent developments towards establishing the structural and biochemical bases of HIV inhibition by, and viral countermeasures of, the restriction factors TRIM5, MxB, APOBEC3, SAMHD1, and BST2/tetherin.
Topics: Animals; DNA, Viral; HIV; Humans; Immune Evasion; Mutation; Reverse Transcription
PubMed: 25939065
DOI: 10.1016/j.sbi.2015.04.004 -
Journal of Translational Medicine Feb 2021COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust...
BACKGROUND
COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.
METHODS
We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.
RESULTS
The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.
CONCLUSIONS
The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.
Topics: Base Sequence; COVID-19; COVID-19 Testing; CRISPR-Cas Systems; Humans; Limit of Detection; RNA, Viral; Real-Time Polymerase Chain Reaction; Reference Standards; Reverse Transcription; SARS-CoV-2
PubMed: 33593370
DOI: 10.1186/s12967-021-02741-5 -
Retrovirology Sep 2021The viral capsid plays a key role in HIV-1 reverse transcription. Recent studies have demonstrated that the small molecule IP6 dramatically enhances reverse... (Review)
Review
The viral capsid plays a key role in HIV-1 reverse transcription. Recent studies have demonstrated that the small molecule IP6 dramatically enhances reverse transcription in vitro by stabilizing the viral capsid. Reverse transcription results in marked changes in the biophysical properties of the capsid, ultimately resulting in its breakage and disassembly. Here we review the research leading to these advances and describe hypotheses for capsid-dependent HIV-1 reverse transcription and a model for reverse transcription-primed HIV-1 uncoating.
Topics: Animals; Capsid; Capsid Proteins; HIV Infections; HIV-1; Humans; Reverse Transcription
PubMed: 34563203
DOI: 10.1186/s12977-021-00566-0 -
Microbiology Spectrum Apr 2015The enzyme reverse transcriptase (RT) was discovered in retroviruses almost 50 years ago. The demonstration that other types of viruses, and what are now called... (Review)
Review
The enzyme reverse transcriptase (RT) was discovered in retroviruses almost 50 years ago. The demonstration that other types of viruses, and what are now called retrotransposons, also replicated using an enzyme that could copy RNA into DNA came a few years later. The intensity of the research in both the process of reverse transcription and the enzyme RT was greatly stimulated by the recognition, in the mid-1980s, that human immunodeficiency virus (HIV) was a retrovirus and by the fact that the first successful anti-HIV drug, azidothymidine (AZT), is a substrate for RT. Although AZT monotherapy is a thing of the past, the most commonly prescribed, and most successful, combination therapies still involve one or both of the two major classes of anti-RT drugs. Although the basic mechanics of reverse transcription were worked out many years ago, and the first high-resolution structures of HIV RT are now more than 20 years old, we still have much to learn, particularly about the roles played by the host and viral factors that make the process of reverse transcription much more efficient in the cell than in the test tube. Moreover, we are only now beginning to understand how various host factors that are part of the innate immunity system interact with the process of reverse transcription to protect the host-cell genome, the host cell, and the whole host, from retroviral infection, and from unwanted retrotransposition.
Topics: Humans; RNA-Directed DNA Polymerase; Retroelements; Retroviridae; Reverse Transcription
PubMed: 26104704
DOI: 10.1128/microbiolspec.MDNA3-0027-2014 -
ELife Oct 2021Circular RNAs (circRNAs) act through multiple mechanisms via their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear...
Circular RNAs (circRNAs) act through multiple mechanisms via their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a challenge, which hinders a comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. circFL-seq benefits from rolling circles and long-read sequencing, and the results showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to those for short-read RNA sequencing. The concordance of the RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of fusion circRNAs at the omics scale may further expand the application of circFL-seq. Taken together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs.
Topics: Cell Line; Humans; Nanopore Sequencing; Protein Isoforms; RNA, Circular; RNA-Seq; Reverse Transcription
PubMed: 34647522
DOI: 10.7554/eLife.69457 -
The EMBO Journal Jan 2021In order to replicate, human immunodeficiency virus (HIV-1) reverse-transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These...
In order to replicate, human immunodeficiency virus (HIV-1) reverse-transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These two key events of the viral life cycle are commonly viewed as separate not only in time, but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early viral replication cycle in macrophages, the natural non-dividing target cells that constitute reservoirs of HIV-1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA (vDNA) and viral RNA, in which multiple viral genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6, and localize to nuclear speckles. Surprisingly, these viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where reverse transcription is pharmacologically suppressed and in untreated cells. We demonstrate that following temporary inhibition, reverse transcription can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear reverse transcription can result in transcription-competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle and may have implications for addressing HIV-1 persistence.
Topics: Active Transport, Cell Nucleus; Cell Line; Cell Nucleus; Cluster Analysis; Cytoplasm; DNA, Viral; Genome, Viral; HEK293 Cells; HIV Infections; HIV-1; Humans; Macrophages; RNA, Viral; Reverse Transcription; THP-1 Cells; Virus Replication
PubMed: 33270250
DOI: 10.15252/embj.2020105247 -
RNA (New York, N.Y.) Jul 2023RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the... (Review)
Review
RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the RNA molecule during the reverse transcription reaction. The resulting cDNA pool is often wrongfully assumed to be quantitatively and molecularly similar to the original RNA input. Sadly, biases and artifacts confound the resulting cDNA mixture. These issues are often overlooked or ignored in the literature by those that rely on the reverse transcription process. In this review, we confront the reader with intra- and intersample biases and artifacts caused by the reverse transcription reaction during RNA sequencing experiments. To fight the reader's despair, we also provide solutions to most issues and inform on good RNA sequencing practices. We hope the reader can use this review to their advantage, thereby contributing to scientifically sound RNA studies.
Topics: Reverse Transcription; DNA, Complementary; Artifacts; RNA; Sequence Analysis, RNA; Bias
PubMed: 36990512
DOI: 10.1261/rna.079623.123 -
Nature Reviews. Microbiology Sep 2019Mammalian cells express a variety of innate immune proteins - known as restriction factors - which defend against invading retroviruses such as HIV-1. Two members of the... (Review)
Review
Mammalian cells express a variety of innate immune proteins - known as restriction factors - which defend against invading retroviruses such as HIV-1. Two members of the tripartite motif protein family - TRIM5α and TRIMCyp - were identified in 2004 as restriction factors that recognize and inactivate the capsid shell that surrounds and protects the incoming retroviral core. Research on these TRIM5 proteins has uncovered a novel mode of non-self recognition that protects against cross-species transmission of retroviruses. Our developing understanding of the mechanism of TRIM5 restriction underscores the concept that core uncoating and reverse transcription of the viral genome are coordinated processes rather than discrete steps of the post-entry pathway of retrovirus replication. In this Review, we provide an overview of the current state of knowledge of the molecular mechanism of TRIM5-mediated restriction, highlight recent advances and discuss implications for the development of capsid-targeted antiviral therapeutics.
Topics: Antiviral Restriction Factors; HIV-1; Host-Pathogen Interactions; Humans; Immunity, Innate; Immunologic Factors; Reverse Transcription; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Virus Uncoating
PubMed: 31312031
DOI: 10.1038/s41579-019-0225-2