Gotaq 1 step rt qpcr

HCN-2 cells were provided by ATCC (CTRL-10742) and were cultured in DMEM (Euroclone, Milan, Italy) + 10% FBS; 100 U/mL penicillin and 100 μg/mL streptomycin were used as antibiotics. The cells were cultured in a 25 cm² culture flask. The same medium, without FBS, was used as inoculum in the mock-infected cells. Cell cultures were incubated with 1 multiplicity of infection (MOI); the incubator was set at 37 °C and 5% CO₂ for 3 h, after which cells were washed two times with lukewarm PBS and refilled with the growth medium (+ 10% FBS). In order to assess the cytopathic effect, we checked the cells daily using an optical microscope (ZOE™ Fluorescent Cell Imager, Bio-Rad, Hercules, CA, USA). RNA was extracted from mock and infected cells. The protocol used was previously described [25 (link)]. Using single-step real-time PCR (GoTaq^® 1-Step RT-qPCR) (Promega, Fitchburg, WI, USA), viral RNA was quantified on a CFX96 (Bio-Rad, Hercules, CA, USA) using primers against two regions of the nucleocapsid (N1 and N2) gene of SARS-CoV-2 (2019-nCoV CDC qPCR Probe Assay emergency kit; IDT, Coralville, IA, USA). The standard curve was generated after quantification of 2019-nCoV_N Positive Plasmid Control (IDT, Coralville, IA, USA). All procedures were performed in agreement with the GLP guidelines adopted in our laboratory.

RT-qPCR technique was performed according to GoTaq®1-Step RT-qPCR (Promega, USA) kit instructions using LNPs and RNase treated LNP samples. Reverse transcription was done at 37 °C for 15 min then hold for 10 min at 95 °C for reverse transcriptase inactivation and GoTaq® DNA polymerase activation. Primers, 0751F and 592R, were used for amplification of the target. Denaturation was done at 95 °C for 10 s, annealing at 45 °C for 30 s, extension at 68 °C for 30 s for 40 cycles (QuantStudio 12 K Flex, ThermoFisher, USA). After completion of PCR cycle, melt curve was analyzed for integrity checking.

Total RNA from cells was extracted using the RNeasy Plus Mini Kit (QIAGEN, 74134) following the manufacturer’s protocol. Synthesis of cDNA was performed using the Maxima H Minus First Strand cDNA Synthesis Kit (ThermoFisher Scientific, K1652) using random hexamer primers. Quantitative PCRs were performed using 200 nM primers, 1:250 (final) diluted cDNA, and 1X GoTaq qPCR Master Mix (Promega, A6002). Amplifications were performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad) and Bio-Rad CFX Manager 3.1 software. To measure supernatant virions, supernatant was mixed 1:1 with virus lysis buffer [20 mM Tris HCl pH7.4, 300 mM NaCl, 2.5% NP-40, and 40 U/mL RNAaseOUT (ThermoFisher)] for 5 min, then diluted 1:5 with nuclease-free water before 2 μL was used as template in a 10 μL GoTaq 1-Step RT-qPCR (Promega, A6020) reaction [73 (link)]. Primer sequences are listed in Table 1.

100 mg of control and inoculated roots were macerated in liquid nitrogen for RNA extraction. According to the manufacturer’s instructions, the total RNA of the samples was extracted with the RNeasy Plant Mini Kit (Qiagen, São Paulo, Brazil). Total RNA was quantified using the NanoDrop ND-1000 spectrophotometer. RNA was eluted in DEPC-treated water (total amount of 4–10 μg RNA), digested with DNAse, and depleted of ribosomal RNA using the GOTAQ^® 1-STEP RT-qPCR (PROMEGA). Subsequently, a 1% RNAse free, agarose gel was made to analyze the extracted RNA. According to the manufacturer’s protocol, sequencing libraries were prepared using the Whole Transcriptome Analysis kit (Applied Biosystems). Libraries were sequenced on the Illumina platform by Lactad company—in Brazil.

MSCs were cultured in DMEM (Euroclone, Milan, Italy) with 10% FBS medium and 100 U/mL penicillin and 100 μg/mL streptomycin in a 24-well plate. DMEM containing 100 U/mL penicillin and 100 µg/mL streptomycin was used as inoculum in the mock-infected cells. Cell cultures were infected with 1 multiplicity of infection (MOI) and incubated at 37 °C and 5% CO₂. After 3 h, cells were washed two times with lukewarm PBS and refilled with the proper growth medium (10% FBS). Optical microscope observation (ZOE™ Fluorescent Cell Imager, Bio-Rad, Hercules, CA, USA) was performed daily to investigate the cytopathic effect. SARS-CoV-2 RNA was extracted from 24, 48, and 72 hpi supernatant using the Maxwell^® RSC Instrument with Maxwell^® RSC Viral Total Nucleic Acid Purification Kit (Promega, Fitchburg, WI, USA). Viral RNA was reverse transcribed in a single-step RT-qPCR (GoTaq 1-Step RT-qPCR; Promega, Fitchburg, WI, USA) on a CFX96 instrument (Bio-Rad, Hercules, CA, USA) using primers specifically designed to target two regions of the nucleocapsid (N1 and N2) gene of SARS-CoV-2 (2019-nCoV CDC qPCR Probe Assay emergency kit; IDT, Coralville, IA, USA), together with primers for the human RNase P gene. Viral copy quantification was assessed by creating a standard curve from the quantified 2019-nCoV_N-positive Plasmid Control (IDT, Coralville, IA, USA).

For assessment of viral replication at 24 and 48 h post-infection, RNA from cell culture supernatants was extracted by using Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega, Fitchburg, WI, USA) through the Maxwell^® RSC Instrument (Promega, Fitchburg, WI, USA). Viral RNA was quantified as previously described [20 (link)]. Briefly, single-step, real-time, RT-qPCR (GoTaq^® 1-Step RT-qPCR, Promega, Fitchburg, WI, USA) and the 2019-nCoV CDC qPCR Probe Assay kit with probes targeting SARS-CoV-2 nucleocapsid (N) gene and the human RNase P gene as an internal control (IDT, Coralville, IA, USA) were used on a CFX96 instrument (Bio-Rad, Hercules, CA, USA). Absolute viral copy number quantification was performed by generating a standard curve from the quantified 2019-nCoV_N-positive Plasmid Control (IDT, Coralville, IA, USA). A cycle threshold (Ct) value of <40 was considered positive, based on CDC guidelines.

Viral RNA was quantified in the saliva of COVID-19 patients by using the Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega, Fitchburg, WI, USA) with the Maxwell^® RSC Instrument (Promega, Fitchburg, WI, USA), as previously described [51 (link)]. Briefly, single-step, real-time RT-qPCR (GoTaq^® 1-Step RT-qPCR, Promega, Fitchburg, WI, USA) and the 2019-nCoV CDC qPCR Probe Assay kit specifically designed to target two regions of the nucleocapsid gene of SARS-CoV-2 (N1 and N2) were used on a CFX96 instrument (Bio-Rad, Hercules, CA, USA). An absolute viral copy of SARS-CoV-2 N gene quantification was performed by generating a standard curve from the quantified 2019-nCoV_N-positive Plasmid Control (IDT, Coralville, IA, USA). A cycle threshold (Ct) value of <40 was considered positive, based on CDC guidelines.