Reverse transcription (RT) reactions were performed in 50 µL containing 5 µL of 10× SuperScript III buffer (Invitrogen), 5 µL of 50 mM MgCl2, 2.5 µL of random hexamers (50 ng/µL; Invitrogen), 2.5 µL of 50 µM poly-dT15, 2.5 µL of 10 mM deoxynucleoside triphosphates (dNTPs), 1.25 µL of RNAseOUT (40 U/µL; Invitrogen), and 2.5 µL of SuperScript III RT (200 U/µL; Invitrogen) [26] . Key reagents and resources are listed in Table S1 . Reverse transcription was performed with both random hexamers and poly-dT in anticipation of this approach being applied to clinical samples containing long polyadenylated SARS-CoV-2 RNAs, for which the combination of poly-dT plus random hexamers may reduce bias towards reverse transcription of any one region (as can be seen with specific reverse primers), the 5′ end (as would be expected with random hexamers), or the 3′ end (as would be expected with poly-dT).
Superscript 3 buffer
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
SuperScript III buffer is a component used in the reverse transcription process for the conversion of RNA to cDNA. It provides a buffer solution to maintain the optimal pH and ionic conditions required for the enzymatic activity of the SuperScript III Reverse Transcriptase.
Automatically generated - may contain errors
Lab products found in correlation
Random hexamer, by Thermo Fisher Scientific (6 mentions)
Rnaseout, by Thermo Fisher Scientific (6 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (5 mentions)
Mgcl2, by Thermo Fisher Scientific (2 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Illumina (1 mentions)
Poly a polymerase, by Illumina (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Amv rt, by Promega (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
7 protocols using superscript 3 buffer
1
Reverse Transcription Protocol for SARS-CoV-2 RNA
2
Polyadenylation for Efficient HIV Transcript Detection
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Reverse transcription from a linker molecule (which we achieve using polyA polymerase to attach a polyA tail) is necessary for efficient reverse transcription of short, prematurely-terminated HIV transcripts limited to the TAR loop [31 (link)]. Therefore, a polyadenylation step was employed prior to reverse transcription and ddPCR for the TAR region [27 (link), 31 (link)]. Each polyadenylation reaction comprised cellular RNA with 3μL of 10x Superscript III buffer (Invitrogen), 3μL of 50mM MgCl2, 1μL of 10mM ATP (Epicentre), 2μL of 4U/μL poly-A polymerase (Epicentre), and 1μL of 40U/μL RNaseOUT (Invitrogen) in a 20μL reaction. The reaction was incubated at 37μC for 45min prior to addition of RT reaction components, including 1.5μL of 10mM dNTPs (Invitrogen), 1.5μL of 50ng/μL random hexamers (Invitrogen), 1.5μL of 50μM oligo dT15, and 1μL of 200U/μL Superscript III reverse transcriptase (Invitrogen). Reverse transcription was performed on the final 30μL reaction at 25.0°C for 10 min, 50.0°C for 50 min, followed by an inactivation step at 85.0°C for 5 min.
3
Reverse Transcription and ddPCR Profiling of HIV Transcripts
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Dilutions of the supernatant virus standard or in vitro transcribed standard were added to reverse transcription reactions to achieve expected inputs of 1 to 10,000 copies per 5 μL cDNA. Reverse transcription (RT) reactions were performed in 50 μL reaction mixture containing 5 μL of 10× SuperScript III buffer (Invitrogen), 5 μL of 50 mM MgCl2, 2.5 μL of random hexamers (50 ng/μL; Invitrogen), 2.5 μL of 50 μM poly-dT15, 2.5 μL of 10 mM deoxynucleoside triphosphates (dNTPs), 1.25 μL of RNAseOUT (40 U/μL; Invitrogen), and 2.5 μL of SuperScript III RT (200 U/μL; Invitrogen) [1 (link)]. The resultant cDNA was split evenly across triplicate wells for ddPCR and dPCR assayed for HIV transcription profiling assays: TAR, Long LTR, Pol, Nef, PolyA, and Tat-Rev [5 (link)].
4
SARS-CoV-2 RT-ddPCR Assay Validation
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Further validations were performed to determine each assay’s sensitivity to inhibition by “background” cellular RNA, as would be expected in clinical samples containing cells. The virion standard (1000 copies per 5μL RT) was added to RT reactions with or without cellular RNA from A549 cells (lung epithelial cell line) or donor PBMC (both added at 100ng/μl per RT, or 500ng per ddPCR well). RT reactions contained a total of 125μL with 12.5 μL of 10× SuperScript III buffer (Invitrogen), 12.5 μL of 50 mM MgCl2, 6.25 μL of random hexamers (50 ng/μL; Invitrogen), 6.25 μL of 50 μM dT15, 6.25 μL of 10 mM deoxynucleoside triphosphates (dNTPs), 3.125 μL of RNAseOUT (40 U/μL; Invitrogen), and 6.25 μL of SuperScript III RT (200 U/μL; Invitrogen). RT reactions were incubated at 25.0°C for 10 min, 50.0°C for 50 min, followed by an inactivation step at 85.0°C for 5 min. Undiluted cDNA (5 μL) was added to each 20 μL ddPCR reaction and replicate ddPCR reactions were performed for each assay.
5
Reverse Transcription cDNA Generation
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A common RT reaction was used to generate cDNA for all ddPCR assays except TAR [27 (link)]. Each 50μL RT contained cellular RNA, 5μL of 10x Superscript III buffer (Invitrogen, Carlsbad, CA), 5μL of 50mM MgCl2, 2.5μL of 50ng/μl random hexamers (Invitrogen), 2.5μL of 50μM dT15, 2.5μL of 10mM dNTPs, 1.25μL of 40U/μL RNaseOUT (Invitrogen), and 2.5μL of 200U/μL Superscript III RT (Invitrogen). Control RT reactions were performed in parallel with participant samples. A ‘6-assay’ synthetic HIV standard was utilized as a positive control [27 (link)]. HIV- donor PBMCs and water that was subjected to nucleic extraction by TRI Reagent served as negative controls for each transcript. The thermocycling conditions were as follows: 25.0°C for 10min, 50.0°C for 50min, followed by an inactivation step at 85.0°C for 5min.
6
Unbiased HIV-2 RNA Quantification
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To mitigate bias towards reverse transcription of the 3’ end (as expected with poly-dT alone), the 5’ end (as expected with random hexamers), or any one region (as expected with specific reverse primers), reverse transcription (RT) reactions were primed using both random hexamers and poly-dT. RT reactions were performed in a 50 μL mixture containing 5 μL of 10× SuperScript III buffer (Invitrogen), 5 μL of 50 mM MgCl2, 2.5 μL of random hexamers (50 ng/μL; Invitrogen), 2.5 μL of 50 μM poly-dT15, 2.5 μL of 10 mM deoxynucleoside triphosphates (dNTPs), 1.25 μL of RNAseOUT (40 U/μL; Invitrogen), 2.5 μL of SuperScript III RT (200 U/μL; Invitrogen), and water or one of the HIV-2 standards +/- cellular RNA from uninfected donor cells. Reactions were performed in a standard thermocycler with the following cycling conditions: 25°C for 10 min, 50°C for 50 min, and an inactivation step at 85°C for 5 min. Aliquots of cDNA from the RT reaction were then added to ddPCR reactions containing primers and probe specific for a given HIV-2 RNA region.
7
RNA Extraction and cDNA Synthesis from Plasma
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The QIAamp Viral RNA Mini Kit (Qiagen, Manchester, UK) was used to extract RNA from 280 µl of plasma according to the manufacturer's instructions. Care was taken to avoid vigorous vortexing to prevent shearing of long RNA templates. The nucleic acids were eluted into 60 µl of 5mM Tris-HCl, pH8.0 (Tris buffer). For the cDNA step (modified from Fan and colleagues, 2006) 10.6 µl of RNA template was added to 9.4 µl reverse transcription mix consisting of 1x SuperScript ® III buffer (Life technologies TM , Invitrogen, Paisley, UK), 0.01 M DTT, 1 µM NS3plaOA primer (Table S1), 0.5 mM dNTPs (Invitrogen), 20 units of RNasin ribonuclease inhibitor, 200 units of SuperScript ® III RT enzyme and 5 units AMV RT (Promega, Southampton, UK). The reaction was performed by incubation at 50 o C for 75 min, followed by heating at 70 o C for 15 min.
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