RNA was isolated from the cells by Trizol (Ambion) and converted to cDNA using reverse transcriptase PCR (cloned AMV reverse transcriptase, Invitrogen). The mRNA was then quantified by real-time RT-PCR using StepOnePlus (Applied Biosystems). Specific primers are included in the supplementary information (Supplementary Table I ). Fast SYBR Green Master Mix (Applied Biosystems) was used to detect fluorescence. Relative quantification was calculated according to the comparative Ct method with normalization to GAPDH. Unless otherwise noted, fold change with normalization to control is shown in the figures.
Cloned amv reverse transcriptase
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Cloned AMV reverse transcriptase is an enzyme used to synthesize complementary DNA (cDNA) from RNA templates. It catalyzes the conversion of single-stranded RNA into double-stranded cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase, by Thermo Fisher Scientific (2 mentions)
Bst 2.0 dna polymerase, by New England Biolabs (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Takara ex taq rt pcr version 2.1 kit, by Takara Bio (1 mentions)
Dna engine opticon 2 continuous fluorescence detection system, by Bio-Rad (1 mentions)
Trizol total rna isolation kit, by Sangon (1 mentions)
Rnase free water, by Merck Group (1 mentions)
Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions)
7 protocols using cloned amv reverse transcriptase
1
Quantitative Gene Expression Analysis
2
RT-PCR Assay for Molecular Detection
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RT-PCR assay was carried out in a total reaction volume10 μl containing 1 μl of 10X PCR buffer, 1 μM each of primers B3 and F3, 2 mM of MgSO4, 1.5 mM of dNTP, 1 mM of DTT, 5 U of RNase inhibitor, 5 U of cloned AMV reverse transcriptase (Invitrogen), 2.5 U of Taq DNA polymerase, 10 μg of template RNA and RNase free deionized water. The reactions were incubated at 60 °C for 30 min, followed by initial denaturation at 94 °C for 4 min, 35 cycles of 94 °C for 45 s, 60 °C for 45 s and 72 °C for 45 s and a final extension at 72 °C for 5 min. RT-PCR procedure was carried out using an automated thermal cycler (Techgene, Germany) and all PCR products were analyzed by 1% agarose gel electrophoresis.
3
RT-PCR detection of COVID-19 from clinical samples
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The total RNA was obtained from the clinical and respiratory samples taken from patients with COVID-19 at Chamran Hospital, extracted using RNX reagent according to the manufacturer’s instruction, and tested using the real-time RT-PCR method (Pishtaz Teb Diagnostics, Iran). For this, RT-PCR assay was performed in a total reaction volume of 10 μl containing 1 μl of 10×PCR buffer, 2 mM of MgSO4, 1.5 mM of dNTP, 1 mM of DTT, 1 μM of each of the B3 and F3 primers, 5 U of RNase inhibitor (Invitrogen), 5 U of cloned AMV reverse transcriptase (Invitrogen), 2.5 U of Taq DNA polymerase, 1.0 μg of the total RNA and RNase-free deionized water. The reactions were first incubated at 60°C for 60 min and then, the following steps were carried out: initial denaturation at 95°C for 5 min, 30 cycles of 95°C for 60 s, 60°C for 60 s, and 72°C for 60 s, and a final extension at 72°C for 10 min. The PCR reaction was done by an automated thermal cycler (Techgene, Germany). PCR products were electrophoresed using 1% agarose gel electrophoresis.
4
Quantification of hSETD1A mRNA Expression
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Total RNA was isolated by using a UNIQ-10 column and TRIzol Total RNA Isolation Kit (Sangon, Shanghai, China). One microgram of total RNA was used for reverse transcription in a reaction volume of 20 μl using Cloned AMV Reverse Transcriptase (Invitrogen). Two microliters of cDNA was used for real-time PCR using TaKaRa Ex Taq RT-PCR Version 2.1 kit (TaKaRa, Shiga, Japan). Gene-specific PCR primers for hSETD1A and GAPDH are listed in Table 2 , and PCR signals were detected with a DNA Engine Opticon 2 Continuous Fluorescence Detection System (Bio-Rad, Hercules, CA, USA). PCR was monitored for 45 cycles using an annealing temperature of 60°C. At the end of the PCR cycles, melt curve analysis and 2% agar electrophoresis were performed to assess the purity of the PCR products. Negative control reactions (no template) were routinely included to monitor potential contamination of reagents. Relative amounts of hSETD1A mRNA were normalized to that of GAPDH mRNA.
5
RT-LAMP Assay for CVB3 Detection
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The CVB3 RT-LAMP assay was carried out in a total 12.5 μl-volume reaction containing 0.8 μM of the inner primers FIP and BIP, 0.1 μM of the outer primers F3 and B3, 1.4 mM dNTPs, 1.25 μl of 10X isothermal amplification buffer, 4 mM of MgSO4, 0.8 M of betaine, 4 U of Bst 2.0 DNA polymerase (New England Biolabs), 5 U of cloned AMV reverse transcriptase (Invitrogen), 1.25 μl of DTT (0.1 M), 0.4 μl of RNasin and 10 μg of target RNA. The mixture was incubated at 60 °C for 90 min and then heated at 80 °C for 10 min to stop of the reaction.
6
SARS-CoV-2 RT-LAMP Detection Protocol
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The RT-LAMP detection of SARS-CoV-2 was performed in a total reaction volume of 12.5 μl containing 1.25 μl of 10× isothermal amplification buffer, 4 mM of MgSO4, 1.4 mM dNTPs, 0.8 M of betaine, 0.1 μM of the outer primers F3 and B3, 0.8 μM of the inner primers FIP and BIP, 4 U of Bst 2.0 DNA polymerase (New England Biolabs), 5 U of cloned AMV reverse transcriptase (Invitrogen), 1.25 μl of DTT (0.1 M), 0.4 μl of RNasin and 60 ng of the total RNA. Finally, 1 μl of SYBR Green I (1/10 dilution in DMSO) was added into the microtube lid, and the mixture was incubated at 65°C for 60 min.
7
Cardiomyocyte mRNA Isolation and Reverse Transcription
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mRNA was isolated directly from cardiomyocytes using Oligo-dT25 Dyna beads® (Invitrogen). The mRNA-bound beads were washed in a Tris buffer solution containing lithium dodecyl sulfate (LiDS) or without LiDS (Buffer A: 10 mM TrisHCl (pH 7.5); 0.15 M LiCl; 1 mM EDTA; 0.1% LiDS; Buffer B: 10 mM TrisHCl (pH 7.5); 0.15 M LiCl; 1 mM EDTA). The beads were resuspended in RNase-free water (Sigma, Dorset, UK), and the mRNA was reverse transcribed using cloned AMV reverse transcriptase (Invitrogen). The resulting cDNA was stored at -80 °C until necessary.
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