Total RNA was extracted and purified with RNAzol RT (cat. no. 888-841-0900; Molecular Research Center) before being reverse transcribed to cDNA with qScript cDNA SuperMix (157031; Quanta Biosciences). PCR was conducted with PrecisionFAST qPCR Master Mix (FASR-LR-SY; Primerdesign Ltd, U.K.) with use of Applied Biosystems StepOnePlus Real-Time PCR System (Life Technologies). The expression levels of respective target genes were normalized to GAPDH, and relative gene expressions were calculated using standard 2−ΔΔCT method. Primers used in this study are listed in Table 1 .
Precisionfast qpcr master mix
Manufactured by Primerdesign
Sourced in United Kingdom
PrecisionFast qPCR Master Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) applications. It contains all the necessary components for efficient and accurate qPCR, including a hot-start DNA polymerase, dNTPs, and buffer.
Automatically generated - may contain errors
Lab products found in correlation
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (6 mentions)
Qiacube ht system, by Qiagen (4 mentions)
Qiaamp 96 virus qiacube ht kit, by Qiagen (4 mentions)
Qscript cdna supermix, by Quanta Biosciences (2 mentions)
Ariamx real time pcr machine, by Agilent Technologies (2 mentions)
Primer express software version 3, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Applied biosystems steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Rnasimple total rna kit, by Tiangen Biotech (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rnazol rt, by Molecular Research Center (1 mentions)
Steponeplus q pcr detection system, by Thermo Fisher Scientific (1 mentions)
13 protocols using precisionfast qpcr master mix
1
Gene Expression Analysis of RNA Samples
2
Quantifying Gene Expression via RT-qPCR
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Taqman RT-qPCR was performed to measure and quantify gene expression. Primers and probe sequences were designed using Primer Express Software Version 3.0.1 (Applied Biosystems Inc., Foster City, CA, USA https://www.thermofisher.com/uk/en/home/technical-resources/software-downloads/primer-express-software-download.html ) (Supplementary Table 1 ). Precision FAST qPCR Master Mix (Primerdesign, Camberley, UK) and AriaMx Real-Time PCR machine (Agilent Technologies) were used to performed RT-qPCR. Each reaction included 3 µl of cDNA, 6.5 µl of master mix, 0.4 µl of forward primer (10 µM), 0.4 µl of reverse primer (10 µM), 0.25 µl of probe (10 µM) and 2.45 RNase-free water. The RT-qPCR conditions comprised a preliminary cycle of 95 °C for 2 min followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. The target gene expression was normalized with human GAPDH or murine β-actin and gene expression was calculated using the relative standard curve method29 (link).
3
SARS-CoV-2 RNA Detection by rRT-PCR
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For viral RNA extraction, briefly, 200 μL of sample was extracted with the QIAamp 96 Virus QIAcube HT kit (QIAGEN) on the QIAcube HT System (QIAGEN) according to manufacturer’s instructions. Purified nucleic acid was then immediately converted to cDNA by reverse transcription with random hexamers using the SensiFAST cDNA Synthesis Kit (Bioline Reagents) as per manufacturer’s instructions. cDNA was used immediately in the real-time reverse transcription PCR (rRT-PCR) or stored at –20°C. A total of 3 μL of cDNA was added to a commercial real-time PCR master mix (PrecisionFast qPCR Master Mix; Primer Design) in a 20 μL reaction mix containing primers and probe (final concentration of 0.9 mM primer and 0.2 mM probe, respectively). Samples were tested for the presence of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes using previously described primers and probes (44 (link), 45 (link)). Thermal cycling and rRT-PCR analyses for all assays were performed on the ABI 7500 FAST real-time PCR system (Applied Biosystems) with the following thermal cycling profile: 95°C for 2 minutes, followed by 45 PCR cycles of 95°C for 5 seconds and 60°C for 25 seconds for N gene and 95°C for 2 minutes, followed by 45 PCR cycles of 95°C for 5 seconds and 55°C for 25 seconds for RdRp/Hel gene and S gene.
4
Quantitative PCR Analysis of Gene Expression
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RNA extraction was performed as per the Qiagen RNeasy kit (Qiagen, Hilden Germany) and 1 μg converted to cDNA using Nanoscript 2 reverse transcriptase (Primer Design Ltd., Southampton, United Kingdom). A master made up of 100 nM of forward and reverse primers (Table 1 ), precisionFAST qPCR Master Mix (Primer Design Ltd., Southampton, United Kingdom) and 5 μl cDNA. Relative levels were normalized to 18s and compared using the ΔΔCt method to obtain fold change relative to control as previously described (Henderson et al., 2019a (link)).
5
SARS-CoV-2 Detection by rRT-PCR
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For viral RNA extraction, 200 μL of sample was extracted with the QIAamp 96 Virus QIAcube HT kit (Qiagen, Germany) on the QIAcube HT System (Qiagen) according to manufacturer’s instructions. Purified nucleic acid was then immediately converted to cDNA by reverse transcription with random hexamers using the SensiFAST cDNA Synthesis Kit (Bioline Reagents, UK) according to manufacturer’s instructions. cDNA was used immediately in the rRT-PCR or stored at -20°C. Three microlitres of cDNA was added to a commercial real-time PCR master mix (PrecisionFast qPCR Master Mix; Primer Design, UK) in a 20 μL reaction mix containing primers and probe with a final concentration of 0.9 mM and 0.2 mM for each primer and the probe, respectively. Samples were tested for the presence of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes using previously described primers and probes (Chan et al., 2020 (link); Corman et al., 2020 (link)). Thermal cycling and rRT-PCR analyses for all assays were performed on the ABI 7500 FAST real-time PCR system (Applied Biosystems, USA) with the following thermal cycling profile: 95C for 2 min, followed by 45 PCR cycles of 95C for 5 s and 60C for 25 s for N gene and 95C for 2 min, followed by 45 PCR cycles of 95C for 5 s and 55C for 25 s for RdRP/Helicase gene and S gene.
6
Isolating and Analyzing Mouse Lung RNA
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Mouse lung tissue was harvested into TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and homogenized immediately. Total cellular RNA was then isolated using the RNAsimple Total RNA Kit (TIANGEN Biotech, Beijing, China) following the manufacturer’s instructions. The quality of RNA was assessed spectrophotometrically by ascertaining that the ratios of A260/280 and A260/230 were higher than 2.
Two µg RNA was used as template to synthesize cDNA using M-MLV Reverse Transcriptase (PROMEGA, Madison, WI, USA) following the manufacturer’s instructions. Real-time PCR was performed using the Precision FAST qPCR Master Mix (Primerdesign Ltd., Cambridge, UK).
The primer sequences used for the genes are:
Two µg RNA was used as template to synthesize cDNA using M-MLV Reverse Transcriptase (PROMEGA, Madison, WI, USA) following the manufacturer’s instructions. Real-time PCR was performed using the Precision FAST qPCR Master Mix (Primerdesign Ltd., Cambridge, UK).
The primer sequences used for the genes are:
7
Quantitative Gene Expression Analysis
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Total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany), with the use of QIAshredder spin column for homogenization and an on-column DNase digestion. Using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), 1 µg of the total RNA was reversely transcribed. The cDNA obtained was analyzed quantitatively using PrecisionFAST qPCR Master Mix (PrimerDesign, Southampton, UK) on an ABI 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primers used are listed in Table S1 . Ct values were generated using default analysis settings and the housekeeping gene GAPDH was used as an internal control. Relative quantification (RQ) was calculated using the 2 −ΔΔCT method.
8
SARS-CoV-2 Detection via rRT-PCR Targeting Multiple Genes
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Three microlitres of cDNA was added to a commercial real-time PCR master mix (PrecisionFast qPCR Master Mix; Primer Design, UK) in a 20 μL reaction mix containing primers and probe with a final concentration of 0.9 μM and 0.2 μM for each primer and the probe, respectively.
Primary screening of pooled samples was performed with a SARS-CoV-2 rRT-PCR targeting the RdRp gene.5 (link) RdRp-positive pools were then ‘deconstructed’, with each individual sample within that positive pool undergoing nucleic acid extraction and testing with rRT-PCRs targeting the E and N genes for confirmation.1 (link) SARS-CoV-2 was reported as ‘detected’ in an individual sample if either the E or N gene was detected, as the RdRp gene detection in the pool could then be attributed to that sample, thereby fulfilling the Australian Public Health Laboratory Network recommendation of two targets for the detection of SARS-CoV-2.6
An in-house positive extraction control, a negative control and a positive control were included with each PCR run. Thermal cycling and rRT-PCR analyses for all assays were performed on the ABI 7500 FAST real-time PCR system (Applied Biosystems, USA) with the following thermal cycling profile: 95°C for 2 min, followed by 45 PCR cycles of 95°C for 5 s and 60°C for 25 s.
Primary screening of pooled samples was performed with a SARS-CoV-2 rRT-PCR targeting the RdRp gene.5 (link) RdRp-positive pools were then ‘deconstructed’, with each individual sample within that positive pool undergoing nucleic acid extraction and testing with rRT-PCRs targeting the E and N genes for confirmation.1 (link) SARS-CoV-2 was reported as ‘detected’ in an individual sample if either the E or N gene was detected, as the RdRp gene detection in the pool could then be attributed to that sample, thereby fulfilling the Australian Public Health Laboratory Network recommendation of two targets for the detection of SARS-CoV-2.6
An in-house positive extraction control, a negative control and a positive control were included with each PCR run. Thermal cycling and rRT-PCR analyses for all assays were performed on the ABI 7500 FAST real-time PCR system (Applied Biosystems, USA) with the following thermal cycling profile: 95°C for 2 min, followed by 45 PCR cycles of 95°C for 5 s and 60°C for 25 s.
9
RNA Expression Analysis Protocol
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Total RNA was extracted and purified with RNAzol RT (catalog no. 888-841-0900, Molecular Research Center) before being reverse-transcribed to cDNA with qScript cDNA SuperMix (157031, Quanta Biosciences). Polymerase chain reaction (PCR) was conducted with PrecisionFAST qPCR Master Mix (FASR-LR-SY, Primerdesign Ltd., UK) with use of Applied Biosystems StepOnePlus Real-Time PCR System (Life Technologies). The expression levels of respective target genes were normalized to GAPDH, and relative gene expressions were calculated using standard 2−ΔΔCT method. Primers used in this study are listed in table S1.
10
qPCR Protocol for Transcript Quantification
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Quantitative polymerase chain reaction (qPCR) was undertaken based on a previously reported technique [42 (link), 43 (link)]. Briefly, reactions were prepared containing, PrecisionFAST qPCR mastermix (Primer Design, Eastleigh, UK), forward primer, z-tagged reverse primer (Table 1 ), Uniprimer probe (Intergen Inc., Oxford, UK), molecular biology grade water and sample cDNA and were run on a StepOne Plus qPCR detection system (Life Technologies, Paisley, UK). Reaction conditions were; initial 95° C for 15 minutes followed by 100 cycles of 95° C for 15 seconds, 55° C for 35 seconds and 72° C for 20 seconds. Samples were run simultaneously with a standard of known transcript copy number, allowing calculation of relative transcript expression. Quantification of GAPDH expression was subsequently used to normalize samples.
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