285 TRANSCRIPT LEVEL OF Hsp70 AND IGF2R GENES IN BOVINE INDIVIDUAL BLASTOCYSTS DERIVED FROM OOCYTES MATURED WITH FBS OR FATTY ACID-FREE BSA

2007 ◽  
Vol 19 (1) ◽  
pp. 258
Author(s):  
E. Warzych ◽  
E. Pers ◽  
A. Buszka ◽  
T. Strabel ◽  
D. Lechniak
Keyword(s):  
Fatty Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Embryo Quality ◽  
Rna Extraction ◽  
Transcript Level ◽  
Large Variability ◽  
Protein Supplements ◽  
Relative Standard

The efficiency of in vitro embryo production in cattle varies between 30 and 40% of blastocysts derived from oocytes matured in vitro. Despite a rigorous selection, some embryos at blastocyst stage displaying normal morphology are not competent to develop after hatching (Maddox-Hyttel et al. 2003 Reproduction (Suppl. 61), 103–116). Therefore, a lot of attention has been focused on embryo quality. Supplements to culture media are one of the factors significantly contributing to this phenomenon. Serum (FBS) and albumin (fatty acid-free BSA, fafBSA) are widely used protein supplements; however, their effect on embryo quality is still variable (Rizos et al. 2003 Biol. Reprod. 68, 236–243). The aim of the present study was to investigate whether good-quality blastocysts (hatched or expanded) derived from oocytes matured in media supplemented with FBS or fafBSA differ in transcript level of 2 genes: heat shock protein (Hsp70) and receptor for insulin-like 2 factor (IGF2R). Bovine Day 8 blastocysts were produced in vitro from oocytes aspirated from slaughterhouse ovaries after maturation in TCM-199 medium supplemented with 10% FBS or 6% fafBSA, as previously described (Makarevich and Markkula 2002 Biol. Reprod. 66, 386–392). On Day 8 post-insemination (pi), good-morphology blastocysts were allocated into 3 groups: (1) hatched, (2) expanded of excellent quality, and (3) expanded of good quality, and individually frozen in liquid nitrogen. Each embryo was processed individually through RNA extraction and cDNA synthesis. Transcript quantitation protocol included: real-time PCR with SYBR Green I, β-actin gene as an internal standard, and relative standard curve method. Data analysis was performed by 2-way ANOVA. In each reaction, an equivalent of 0.125 embryo (2.5 �L of cDNA) was used, and 43 blastocysts were analyzed. All analyzed embryos were positive for the Hsp70 transcript, whereas IGF2R mRNA was detected in only 58% of blastocysts regardless of the maturation medium. A large variation in relative abundance (RA) was observed among individual embryos: coefficients of variation were 114.5 and 323.6% for IGF2R and Hsp70, respectively. Due to the distribution of Hsp70 RA, log transformation was performed. Real-time PCR data revealed a maximum 100-fold variation for the reference gene. Hatched blastocysts were characterized by a significantly lower RA for both analyzed genes. The 2 classes of expanded blastocysts did not differ in transcript level. With regard to protein supplements, only the RA for Hsp70 gene was significantly affected. This transcript was more abundant in embryos derived from fafBSA-supplemented IVM medium. The present results confirmed previously the described phenomenon concerning a large variability in mRNA content in single pre-implantation embryos. Moreover, because embryos able to hatch significantly differed in RA from their expanded counterparts, it is possible to relate embryo quality to transcript level.

262 ASSESSMENT OF HSP70-1 TRANSCRIPTION LEVELS IN IMMATURE OOCYTES FROM BOS TAURUS AND BOS INDICUS COWS RAISED IN A TROPICAL CLIMATE

2007 ◽  
Vol 19 (1) ◽  
pp. 247
Author(s):  
L. S. A. Camargo ◽  
J. H. M Viana ◽  
R.V. Serapião ◽  
M. F. M. Guimarães ◽  
W. F. Sá ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Real Time ◽  
Real Time Pcr ◽  
Bos Taurus ◽  
Rna Extraction ◽  
Bos Indicus ◽  
Tropical Climate ◽  
Relative Quantification ◽  
Vitro Fertilization

Heat stress is one of the main causes of low conception rate in Bos taurus cows in a tropical climate. On the other hand, in this environment, oocytes from Bos indicus show greater developmental capacity after in vitro fertilization than those from Bos taurus, suggesting an adaptation to the hot climate. Heat shock proteins (HSP) are chaperones that promote protection against heat damage, and their transcription is associated to stress. The aim of this study was to evaluate the expression of HSP70-1 gene (Genbank NM174550), a member of HSP family, in oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) cows raised in the tropical climate located at 21�35′′S latitude, 43�51′′W longitude, and 435 m altitude. Cumulus–oocyte complexes were recovered by oocyte pickup from mature non-lactating Holstein (n = 4) and Gyr (n = 4) donor cows during the hot season. Cumulus cells of viable oocytes were removed by vortexing in TALP-HEPES plus BSA, and pools (3 for each breed) with 12 immature oocytes were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction. Total RNA extraction was performed using Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and first strands were synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000; Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) and cDNA equivalent to 1.2 oocytes and gene specific primers. Expression of the GAPDH gene was used as endogenous reference. Calculations of relative quantification were performed by the comparative Ct method, using the lowest value found in Bos indicus oocytes as calibrator; values (mean � SE) are shown as n-fold difference relative to the calibrator. Statistical comparison between breeds was performed by analysis of variance. Oocytes from Holstein cows showed a higher level (P < 0.05) of HSP70-1 expression (1.82 � 0.22) than oocytes recovered from Gyr cows (1.12 � 0.11). Previous study reported that oocytes from Gyr cows in a tropical climate showed a higher blastocyst rate after in vitro fertilization than Holstein oocytes (Camargo et al. 2006 Reprod. Fertil. Dev. 18, 243 abst). The lower level of HSP70-1 in Gyr oocytes suggests that they were less subject to stress than the Holstein ones, which may reflect their capacity to develop after fertilization. This effect may be, at least in part, due to the ability of Bos indicus cows to regulate body temperature in a hot environment, causing less stress on oocytes. Financial support was provided by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogen�tica, Vi�osa, Brazil, for the real-time PCR machine.

280 EXPRESSION OF HSP70.1 GENE IN IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN CR2 MEDIUM SUPPLEMENTED WITH KNOCKOUT™SR

2007 ◽  
Vol 19 (1) ◽  
pp. 255
Author(s):  
R. V. Serapião ◽  
L. S. de Almeida Camargo ◽  
A. de Almeida Ramos ◽  
I. de Moura Folhadella ◽  
J. Polisseni ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Culture Medium ◽  
Rna Extraction ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Relative Quantification ◽  
Bovine Embryos

The exposure of embryos to serum during in vitro culture can affect morphology, metabolism, tolerance to cryopreservation, and expression of specific transcripts. On the other hand, serum-free medium seems to avoid some of those serum effects. KnockoutTMSR (GIBCO Laboratories, Grand Island, NY, USA) is a serum replacer optimized to support embryonic stem cells in culture and can also be used to replace serum during culture of in vitro-fertilized bovine embryos. The expression of genes associated with stress response, such as heat shock proteins (HSP), can be affected by in vitro culture conditions, being easily induced by a variety of stress agents, including culture medium components. This study aimed to determine whether KnockoutSR or serum in culture medium alters the relative abundance of HSP70.1 transcripts in in vitro-fertilized bovine embryos. Cumulus–oocyte complexes obtained from slaughterhouse ovaries were matured and feritlized in vitro. Presumptive zygotes were randomly cultured with their own cumulus cells in CR2aa medium supplemented with 10% fetal calf serum (GIBCO-BRL, Paisley, UK; FCS group), 10% KnockoutSR (GIBCO-BRL; KSR group), or 3 mg mL-1 of polyvinyl alcohol (PVA group). All steps were performed at 38.5�C, under 5% CO2 in air and 95% humidity. Blastocysts on Day 8 post-fertilization were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction (3 replicates for each group). Total RNA extraction was performed using an Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and the first strand was synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000 Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) with cDNA equivalent to 0.8 embryos and gene-specific primers. Expression of the glyceraldehyde 3-phosphate dehydrogenase gene was used as endogenous reference. Calculations of relative quantification were performed by comparative Ct method, using the value found in the PVA group as calibrator. Expression levels for the FCS and KSR groups were 1.2 � 0.06- and 1.4 � 0.08-fold differences relative to the PVA group without differences (P > 0.05). These data show that bovine embryos cultured in medium supplemented with KSR have the same HSP70-1 expression pattern as those in medium with added FCS, suggesting that embryos in both groups are under the same stress conditions. This work was supported by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogenetica, Vi�osa, Brazil, for the real-time PCR machine.

scholarly journals The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2341
Author(s):  
Normann Steiner ◽  
Karin Jöhrer ◽  
Selina Plewan ◽  
Andrea Brunner-Véber ◽  
Georg Göbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

scholarly journals The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Dong Chen ◽  
Yaqin Wang ◽  
Feiya Yang ◽  
Adili Keranmu ◽  
Qingxin Zhao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Bioinformatic Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

scholarly journals Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis

2009 ◽  
Vol 58 (5) ◽  
pp. 648-655 ◽  
Author(s):  
Kristel Lourdault ◽  
Florence Aviat ◽  
Mathieu Picardeau
Keyword(s):  
Guinea Pig ◽  
Real Time ◽  
Real Time Pcr ◽  
Guinea Pigs ◽  
Infection Model ◽  
Avirulent Strain ◽  
Leptospira Interrogans ◽  
Quantitative Real Time Pcr ◽  
Guinea Pig Model

The dynamics of leptospirosis infection have been poorly studied. The purpose of this study was to determine the LD50, rate of bacterial dissemination, histopathology and antibody responses against leptospira following inoculation with the highly virulent Leptospira interrogans Fiocruz L1-130 strain in a guinea pig model of leptospirosis. Three routes of infection (intraperitoneal, conjunctival and subcutaneous inoculation) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in the blood and tissues of infected animals were determined over a 1 week course of infection using quantitative real-time PCR (qPCR). Bacteraemia peaked at day 5 post-infection reaching more than 5×104 leptospires ml−1. The highest spirochaetal load was found in the liver and kidneys, and was associated with alterations in organ tissues and a decline in liver and kidney functions. In contrast, lesions and bacteria were not detected in guinea pigs infected with an avirulent strain derived from a high-passage-number in vitro-passaged variant of the Fiocruz L1-130 strain. The use of qPCR supports the findings of earlier studies and provides an easy and reliable method for the quantification of L. interrogans in the tissues of infected animals. qPCR will be used in future studies to evaluate the efficacy of vaccine candidates against leptospirosis and the virulence of selected L. interrogans mutants relative to the parental strain.

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

2020 ◽  
pp. 15-34
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Tissue Regeneration ◽  
Real Time Pcr ◽  
Molecular Level ◽  
Northern Blotting ◽  
Tissue Constructs ◽  
Low Sensitivity

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

(-)-Epigallocatechin Gallate Promotes MicroRNA 145 Expression against Myocardial Hypoxic Injury through Dab2/Wnt3a/β-catenin

2020 ◽  
Vol 48 (02) ◽  
pp. 341-356
Author(s):  
Chiu-Mei Lin ◽  
Wei-Jen Fang ◽  
Bao-Wei Wang ◽  
Chun-Ming Pan ◽  
Su-Kiat Chua ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Real Time ◽  
Real Time Pcr ◽  
Myocardial Infarct ◽  
Epigallocatechin Gallate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rat Cardiomyocytes

MicroRNA 145 (miR-145) is a critical modulator of cardiovascular diseases. The downregulation of myocardial miR-145 is followed by an increase in disabled-2 (Dab2) expression in cardiomyocytes. (-)-epigallocatechin gallate (EGCG) is a flavonoid that has been evaluated extensively due to its diverse pharmacological properties including anti-inflammatory effects. The aim of this study was to investigate the cardioprotective effects of EGCG under hypoxia-induced stress in vitro and in vivo. The hypoxic insult led to the suppression of miR-145 expression in cultured rat cardiomyocytes in a concentration-dependent manner. Western blotting and real-time PCR were performed. In rat myocardial infarction study, in situ hybridization, and immunofluorescent analyses were adopted. The western blot and real-time PCR data revealed that hypoxic stress with 2.5% O2 suppressed the expression of miR-145 and Wnt3a/[Formula: see text]-catenin in cultured rat cardiomyocytes but augmented Dab2. Treatment with EGCG attenuated Dab2 expression, but increased Wnt3a and [Formula: see text]-catenin in hypoxic cultured cardiomyocytes. Following in vivo myocardial infarction (MI) study, the data revealed the myocardial infarct area reduced by 48.5%, 44.6%, and 48.5% in EGCG (50[Formula: see text]mg/kg) or miR-145 dominant or Dab2 siRNA groups after myocardial infarction for 28 days, respectively. This study demonstrated that EGCG increased miR-145, Wnt3a, and [Formula: see text]-catenin expression but attenuated Dab2 expression. Moreover, EGCG ameliorated myocardial ischemia in vivo. The novel suppressive effect was mediated through the miR-145 and Dab2/Wnt3a/[Formula: see text]-catenin pathways.

scholarly journals Evaluation of RealStar® Alpha Herpesvirus PCR Kit for Detection of HSV-1, HSV-2, and VZV in Clinical Specimens

2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
Author(s):  
Cyril C. Y. Yip ◽  
Siddharth Sridhar ◽  
Kit-Hang Leung ◽  
Andrew K. W. Cheng ◽  
Kwok-Hung Chan ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Test Performance ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Varicella Zoster ◽  
Simplex Virus ◽  
Hsv 1

Several commercial PCR kits are available for detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), but the test performance of one CE-marked in vitro diagnostic kit—RealStar® alpha Herpesvirus PCR Kit—has not been well studied. This study evaluated the performance of RealStar® alpha Herpesvirus PCR Kit 1.0 on the LightCycler® 480 Instrument II for detection and differentiation of HSV-1, HSV-2, and VZV in human clinical specimens. We evaluated the analytical sensitivity of the RealStar® and in-house multiplex real-time PCR assays using serial dilutions of nucleic acids extracted from clinical specimens. The analytical sensitivity of the RealStar® assay was 10, 32, and 100 copies/reaction for HSV-1, HSV-2, and VZV, respectively, which was slightly higher than that of the in-house multiplex real-time PCR assay. Reproducibility of the cycle threshold (Cp) values for each viral target was satisfactory with the intra- and interassay coefficient of variation values below 5% for both assays. One-hundred and fifty-three clinical specimens and 15 proficiency testing samples were used to evaluate the diagnostic performance of RealStar® alpha Herpesvirus PCR Kit against the in-house multiplex real-time PCR assay. The RealStar® assay showed 100% sensitivity and specificity when compared to the in-house assay. Cp values of the RealStar® and in-house assays showed excellent correlation. RealStar® alpha Herpesvirus PCR is a sensitive, specific, and reliable assay for the detection of HSV-1, HSV-2, and VZV, with less extensive verification requirements compared to a laboratory developed assay.

scholarly journals Development and application of a triplex real-time PCR assay for simultaneous detection of avian influenza virus, Newcastle disease virus, and duck Tembusu virus

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Xiyu Zhang ◽  
Ming Yao ◽  
Zhihui Tang ◽  
Daning Xu ◽  
Yan Luo ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Standard Deviation ◽  
Real Time ◽  
Real Time Pcr ◽  
Virus Isolation ◽  
Simultaneous Detection ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Duck Tembusu Virus ◽  
Relative Standard

Abstract Background Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. Results The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won’t cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. Conclusion The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

scholarly journals A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Christian Shema Mugisha ◽  
Hung R. Vuong ◽  
Maritza Puray-Chavez ◽  
Adam L. Bailey ◽  
Julie M. Fox ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Rna Extraction ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Dna Viruses ◽  
Culture Supernatants ◽  
Hiv 1 ◽  
Rna And Dna

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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