DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

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A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)

3 Biotech ◽

10.1007/s13205-020-02514-9 ◽

2020 ◽

Vol 10 (12) ◽

Author(s):

Siti Suriawati Badai ◽

Omar Abd Rasid ◽

Ghulam Kadir Ahmad Parveez ◽

Mat Yunus Abdul Masani

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Oil Palm ◽

Real Time Pcr ◽

Extraction Method ◽

Rna Extraction ◽

Quantitative Real Time Pcr

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Molecular diagnosis of Iraqi chronic myeloid leukemia patients using quantitative real-time PCR

Journal of Biotechnology Research Center ◽

10.24126/jobrc.2010.4.2.125 ◽

2010 ◽

Vol 4 (2) ◽

pp. 64-69

Author(s):

Adeeb Abbas ◽

Majeed A. Sabbah ◽

Abdul-salam Hatam ◽

Luma A. Yasser ◽

Baan Abdul-Latif

Keyword(s):

Bone Marrow ◽

Real Time ◽

Real Time Pcr ◽

Myeloid Leukemia ◽

Rna Extraction ◽

Fusion Transcript ◽

Quantitative Real Time Pcr ◽

Rt Pcr ◽

Chronic Granulocytic Leukemia ◽

Granulocytic Leukemia

hronic myeloid leukemia (CML), also known as chronic granulocytic leukemia, is a form of leukemia characterized by the increased and unregulated growth of myeloid cells in the bone marrow and the accumulation of these cells in peripheral blood. Total RNA extraction, cDNA and quantitative real-time PCR (Q-RT-PCR) were done for thirty CML Iraqi patients. Hematology tests (hemoglobin, platelets and WBCs counts) were done for the same samples in the same time. The results of this study show that some samples with normal hematology values have BCR-ABL (p210) fusion transcript with molecular analysis by Q-RT-PCR. This indicates the importance of this technique in the diagnosis and monitoring the therapy of CML patients.

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More DNA and RNA of HBV SP1 splice variants are detected in genotypes B and C at low viral replication

Scientific Reports ◽

10.1038/s41598-021-03304-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ka-Cheung Luk ◽

Jeffrey Gersch ◽

Barbara J. Harris ◽

Vera Holzmayer ◽

Dora Mbanya ◽

...

Keyword(s):

Viral Load ◽

Real Time ◽

Real Time Pcr ◽

Splice Variant ◽

Splice Variants ◽

Pcr Primers ◽

Quantitative Real Time Pcr ◽

Viral Loads ◽

Hbv Genotypes ◽

Dna And Rna

AbstractHBV produces unspliced and spliced RNAs during replication. Encapsidated spliced RNA is converted into DNA generating defective virions that are detected in plasma and associated with HCC development. Herein we describe a quantitative real-time PCR detection of splice variant SP1 DNA/RNA in HBV plasma. Three PCR primers/probe sets were designed detecting the SP1 variants, unspliced core, or X gene. Plasmids carrying the three regions were constructed for the nine HBV genotypes to evaluate the three sets, which were also tested on DNA/RNA extracted from 193 HBV plasma with unknown HCC status. The assay had an LOD of 80 copies/ml and was equally efficient for detecting all nine genotypes and three targets. In testing 84 specimens for both SP1 DNA (77.4%) and RNA (82.1%), higher viral loads resulted in increased SP1 levels. Most samples yielded < 1% of SP1 DNA, while the average SP1 RNA was 3.29%. At viral load of ≤ 5 log copies/ml, the detectable SP1 DNA varied by genotype, with 70% for B, 33.3% for C, 10.5% for E, 4% for D and 0% for A, suggesting higher levels of splicing in B and C during low replication. At > 5 log, all samples regardless of genotype had detectable SP1 DNA.

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Recruitment of cyanobacteria by reverse transcription quantitative real-time PCR based on expression of Microcystis gene

PeerJ ◽

10.7717/peerj.7188 ◽

2019 ◽

Vol 7 ◽

pp. e7188

Author(s):

Long Yu ◽

Xiaofei Wu ◽

Yang Yu ◽

Limei Shi ◽

Min Zhang

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Lake Taihu ◽

Rna Extraction ◽

Sybr Green ◽

Quantitative Detection ◽

Control Group ◽

Quantitative Real Time Pcr ◽

Total Rna ◽

Pcr Method

In this study, a SYBR Green quantitative real-time PCR method was established and applied. Relative expression of the synthetic genes from Microcystis gas vesicles (gvpC), algal toxin genes (mcyA), and polysaccharides (espL) from water and sediments of Meiliang Bay and from the center of Lake Taihu were tested from January to June, 2017. Indoor Microcystis aeruginosa was used as the control group. The kit for total RNA extraction in Microcystis was optimized. Results showed that the optimized kit extracted high-concentrations and high-quality total RNA from Microcystis. The extraction purity and concentration were significantly higher than those extracted by the original kit. The transcription level of gvpC increased gradually until a peak was reached in March. However, expression of gvpC decreased continuously at the proliferating and floating stages of Cyanobacterial biomass. The maximum level of expression of gvpC in April in comparison to expression of mcyA in March occurred first. We found that the SYBR Green qRT-PCR method, which is characterized by high specificity, repeatability, is rapid, and can be used for quantitative detection of expression of gvpC, mcyA, and espL. The recruitment of cyanobacteria is the process in which cyanobacteria in the sediment began to regain their activity, started to grow and migrated to the water column.

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Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR

PLoS ONE ◽

10.1371/journal.pone.0046451 ◽

2012 ◽

Vol 7 (9) ◽

pp. e46451 ◽

Cited By ~ 176

Author(s):

Deshui Liu ◽

Lindan Shi ◽

Chenggui Han ◽

Jialin Yu ◽

Dawei Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Nicotiana Benthamiana ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Expression Studies ◽

Gene Expression Studies

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KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

BMC Cardiovascular Disorders ◽

10.1186/s12872-021-02053-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yi Wang ◽

Hongjuan Liao ◽

Yueheng Wang ◽

Jinlin Zhou ◽

Feng Wang ◽

...

Keyword(s):

Protein Synthesis ◽

Angiotensin Ii ◽

Cardiac Hypertrophy ◽

Western Blot ◽

Real Time ◽

Real Time Pcr ◽

Mtor Signaling ◽

Heart Weight ◽

Cardiomyocyte Hypertrophy ◽

Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

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