Accuscript high fidelity reverse transcriptase

Manufactured by Agilent Technologies

Sourced in United States

AccuScript High Fidelity Reverse Transcriptase is a reverse transcriptase enzyme used for the conversion of RNA to cDNA. It demonstrates high fidelity and efficiency in the reverse transcription process.

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Lab products found in correlation

Faststart high fidelity taq dna polymerase, by Roche (3 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Superase in rnase inhibitor, by Thermo Fisher Scientific (2 mentions) Race kit, by Thermo Fisher Scientific (2 mentions) Q5 dna polymerase, by New England Biolabs (2 mentions) Miseq platform, by Illumina (2 mentions) Qiaquick pcr purification kit, by Qiagen (2 mentions) Random hexamer, by Thermo Fisher Scientific (1 mentions) Dynamo flash sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions) Plant rna purification reagent, by Thermo Fisher Scientific (1 mentions) Gotaq dna polymerase, by Promega (1 mentions) Escherichia coli dh5α, by Promega (1 mentions) Pfuultra 2 fusion hotstart dna polymerase, by Agilent Technologies (1 mentions) Rnalater, by Thermo Fisher Scientific (1 mentions) Pgem t easy vector, by Promega (1 mentions) Rq1 rnase free dnase, by Promega (1 mentions) Sv total rna isolation system, by Promega (1 mentions) Q5 polymerase, by New England Biolabs (1 mentions) Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions) Qiaamp viral rna mini kit, by Qiagen (1 mentions)

Close spelling variants of this product

AccuScript (6 citations) AccuScript Reverse Transcriptase (2 citations) Reverse transcriptase (2 citations)

9 protocols using accuscript high fidelity reverse transcriptase

HVR1 Amplification and Sequencing for Hepatitis C Virus Genotyping

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HVR1 amplification was performed as described previously^{27 (link)}. In brief, total RNA was extracted from 250 μl of plasma by a modified guanidinium thiocyanate-phenol/chlorophorm method using Trizol (Life Technologies, Carlsbad, CA, USA). RNA was subjected to reverse transcription at 37 °C for 30 minutes using AccuScript High Fidelity Reverse Transcriptase (Agilent Technologies, Santa Clara, CA, USA) and random hexamers (Life Technologies). A gene fragment encompassing HVR1 was amplified in two-step PCR using FastStart High Fidelity Taq DNA Polymerase (Roche, Indianapolis, IN, USA) using target-specific primers (Table 2). Additionally, primers employed in the second round PCR contained tags recognized by GS Junior sequencing platform and standard 10-nucleotide multiplex identifiers^{27 (link)}.Table 2

Primer sequences employed in the study.

	genotype 1b HVR1 amplification	Positions of HCV genome	genotype 3 HVR1 amplification	Positions of HCV genome
First round PCR	Forward: 5′-GGTGCTCACTGGGGAGTCCT-3′	1389–1408	Forward: 5′-ATGGCATGGGATATGAT-3′	1291–1307
First round PCR	Reverse: 5′-CATTGCAGTTCAGGGCCGTGCTA-3′	1632–1610	Reverse: 5′-AAGGCCGTCCTGTTGA-3′	1619–1604
Second round PCR	Forward: 5′- TCCATGGTGGGGAACTGGGC-3′	1428–1447	Forward: 5′-GGCAACTGGGCCAAGGTCGC-3′	1437–1456
Second round PCR	Reverse: 5′-TGCCAACTGCCATTGGTGTT-3′	1603–1584	Reverse: 5′-ATGTGCCACGAGCCATTGGT-3′	1606–1587

Janiak M., Perlejewski K., Grabarczyk P., Kubicka-Russel D., Zagordi O., Berak H., Osuch S., Pawełczyk A., Bukowska-Ośko I., Płoski R., Laskus T, & Caraballo Cortés K. (2019). Hepatitis C virus (HCV) genotype 1b displays higher genetic variability of hypervariable region 1 (HVR1) than genotype 3. Scientific Reports, 9, 12846.

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HVR1 Amplification from Serum RNA

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HVR1 amplification was done as described in a previous publication [20 (link)]. In brief, total RNA was extracted from 250 μl of serum by a modified guanidinium thiocyanate-phenol/chlorophorm method using Trizol (Life Technologies, Carlsbad, CA, USA). Next, RNA was subjected to reverse transcription at 42°C for 60 minutes using AccuScript High Fidelity Reverse Transcriptase (Agilent Technologies, Santa Clara, CA, USA) and random hexamers. A region of 175 nt length encompassing HVR1 was amplified in two-step PCR using FastStart High Fidelity Taq DNA Polymerase (Roche, Indianapolis, IN, USA). Primers used for the first round amplification were as follows: 5′-CATTGCAGTTCAGGGCCGTGCTA-3′ (nt 1632–1610) and 5′-GGTGCTCACTGGGGAGTCCT-3′ (nt 1389–1408), according to the sequence of reference strain H77 (GenBank accession no. AF009606). Primers employed in the second PCR contained tags recognized by GS Junior sequencing platform, standard 10-nucleotide multiplex identifiers and target-complementary sequence [5′- TCCATGGTGGGGAACTGGGC-3′ (positions 1428–1447) and 5′-TGCCAACTGCCATTGGTGTT-3′ (positions 1603–1584)] [20 (link)].

Caraballo Cortes K., Rosińska M., Janiak M., Stępień M., Zagordi O., Perlejewski K., Osuch S., Pawełczyk A., Bukowska-Ośko I., Płoski R., Grabarczyk P., Laskus T, & Radkowski M. (2018). Next-generation sequencing analysis of a cluster of hepatitis C virus infections in a haematology and oncology center. PLoS ONE, 13(3), e0194816.

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Sanger Sequencing of Porcine Deltacoronavirus Genomes

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Attempts at next-generation sequencing using an Illumina MiSeq platform generated minimal coverage, so we sorted to Sanger sequencing using the primer system outlined by Liang et al.^{42 (link)}, with one addition: to obtain the 5′ ends of the viral genomes, a rapid amplification of cDNA ends (RACE) kit was used per the manufacturer’s protocols (Life Technologies), and the resulting amplicons were TA-cloned into plasmids and sequenced. PCR amplicons for Sanger sequencing were amplified using AccuScript High-Fidelity reverse transcriptase (Agilent Technologies) in the presence of SUPERase-In RNase inhibitor (Ambion), followed by PCR with Q5 DNA polymerase (New England Biolabs). They were next purified using a QIAquick PCR purification kit (Qiagen) before TA cloning. The inserts in the plasmids were subsequently sequenced bidirectionally using a gene-walking approach, on the basis of obtaining at least 800 bp or non-ambiguous sequence. Briefly, pairs of non-overlapping primers and Q5 polymerase were used to produce 42 separate amplicons corresponding to the PDCoV genome, and each amplicon was Sanger sequenced bidirectionally.

Lednicky J.A., Tagliamonte M.S., White S.K., Elbadry M.A., Alam M.M., Stephenson C.J., Bonny T.S., Loeb J.C., Telisma T., Chavannes S., Ostrov D.A., Mavian C., Beau De Rochars V.M., Salemi M, & Morris JG J.r. (2021). Independent infections of porcine deltacoronavirus among Haitian children. Nature, 600(7887), 133-137.

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Transcriptional Response to Auxin Inhibitor

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WT seeds were planted in ½ MS liquid medium, stratified at 4 °C for 2 days, and transferred to a rotary shaker under 24 h light conditions. At 4 DPI, cultures were supplemented with AVG (aminoethoxyvinylglycine) to a final concentration of 40 M and incubated for 24 h on the shaker under constant light. A subset of cultures was then treated with 40 M 2,4-D. Whole seedlings from both pools were collected after 4 hours and RNA was extracted using Plant RNA Purification Reagent (Thermo-Fisher Scientific). cDNA was subsequently synthesized using AccuScript High Fidelity Reverse Transcriptase (Agilent). RT-QPCR was performed using a BioRad Real Time Quantitative Thermocycler and the DyNamo Flash SYBR Green qPCR Kit (Thermo-Fisher Scientific). Genes were amplified using the primers listed in Table 1. Amplification efficiency of primers were tested and found to be ≥1.9. Relative transcript abundance was then determined using the delta CT method and normalized to the ADENINE PHOSPHORIBOSYL TRANSFERASE 1 (APT1) gene [71 (link)].

Schuetz M., Fidanza M, & Mattsson J. (2019). Identification of Auxin Response Factor-Encoding Genes Expressed in Distinct Phases of Leaf Vein Development and with Overlapping Functions in Leaf Formation. Plants, 8(7), 242.

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Sanger Sequencing of PDCoV Genomes

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Attempts at next-generation sequencing using an Illumina MiSeq platform generated minimal coverage, so we sorted to Sanger Sequencing using the primer system outlined by Liang et al^{45 (link)}, with one addition: to obtain the 5′ ends of the viral genomes, a Rapid Amplification of cDNA Ends (RACE) kit was used per the manufacturer’s protocols (Life Technologies, Carlsbad, CA, USA), and the resulting amplicons TA-cloned into plasmids and sequenced. PCR amplicons for Sanger Sequencing were amplified using AccuScript High-Fidelity reverse transcriptase (Agilent Technologies, Inc., Santa Clara, CA) in the presence of SUPERase-In RNase inhibitor (Ambion, Austin, TX), followed by PCR with Q5 DNA polymerase (New England Biolabs. They were next purified using a QIAquick PCR purification kit (Qiagen Inc., Germantown, MD) before TA-cloning. The inserts in the plasmids were subsequently sequenced bidirectionally using a gene-walking approach, based on obtaining at least 800 bp or non-ambiguous sequence. Briefly, pairs of non-overlapping primers and Q5 polymerase were used to produce 42 separate amplicons corresponding to the PDCoV genome, and each amplicon was Sanger sequenced bidirectionally.

Lednicky J.A., Tagliamonte M.S., White S.K., Elbadry M.A., Alam M.M., Stephenson C.J., Bonny T.S., Loeb J.C., Telisma T., Chavannes S., Ostrov D.A., Mavian C., Beau De Rochars V.M., Salemi M, & Morris JG J.r. (2021). Emergence of porcine delta-coronavirus pathogenic infections among children in Haiti through independent zoonoses and convergent evolution. medRxiv.

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Molecular analysis of Macrobrachium carcinus

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Macrobrachium carcinus specimens were from the Grijalva River, Centla, Tabasco, Mexico (latitude 18°14′11.9″ N, longitude 92°39′49.4″). All oligonucleotides were purchased from Integrated (DNA Technologies, Inc., Coralville, IA, USA). Escherichia coli DH5α, pGEM-T easy vector, RQ1 RNase-free DNase, SV Total RNA Isolation System, and GoTaq DNA polymerase were purchased from Promega (Madison, WI, USA). Luria-Bertani (LB) agar plates (1% tryptone, 0.5% yeast extract, 1% NaCl, 15 g/L agar, pH 7.0) with 100 µg/mL ampicillin was used for E. coli transformants selection. PfuUltra II Fusion HotStart DNA polymerase and AccuScript High-Fidelity Reverse Transcriptase were from Agilent Technologies (Santa Clara, CA, USA). RNAlater was from Life Technologies (Gaithersburg, MD, USA). All chemicals were of analytical grade and purchased from Sigma–Aldrich Co. (St. Louis, MO, USA) or from Productos Químicos Monterrey (Monterrey, Nuevo León, Mexico).

Viader-Salvadó J.M., Aguilar Briseño J.A., Gallegos-López J.A., Fuentes-Garibay J.A., Alvarez-González C.A, & Guerrero-Olazarán M. (2020). Identification and in silico structural and functional analysis of a trypsin-like protease from shrimp Macrobrachium carcinus. PeerJ, 8, e9030.

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HVR1 Amplification from Serum Samples

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HVR1 amplification was performed from pretreatment serum samples as described previously
[26 (link)]. In brief, viral RNA was extracted from 250 μl of serum by modified guanidinium thiocyanate-phenol/chlorophorm method, then subjected to reverse transcription at 37°C for 30 minutes using AccuScript High Fidelity Reverse Transcriptase (Agilent Technologies). A fragment of E2 region containing HVR1 was amplified in two-step PCR using FastStart High Fidelity Taq DNA Polymerase (Roche). Primers for the second round PCR contained tags recognized by GS Junior sequencing platform, standard 10-nucleotide multiplex identifiers (MID) and target-specific sequence.

Caraballo Cortés K., Zagordi O., Perlejewski K., Laskus T., Maroszek K., Bukowska-Ośko I., Pawełczyk A., Płoski R., Berak H., Horban A, & Radkowski M. (2014). Deep sequencing of hepatitis C virus hypervariable region 1 reveals no correlation between genetic heterogeneity and antiviral treatment outcome. BMC Infectious Diseases, 14, 389.

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SARS-CoV-2 Genome Sequencing Protocol

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Sanger sequencing was performed on vRNA extracted from the stage 4 filter of the PCIS, since this sample had the lowest Cq value. A gene walking approach with non-overlapping primers was used for sequencing (Lednicky et al., 2020b (link)), details of which are provided in the supplementary material. Briefly, cDNA produced using AccuScript high-fidelity reverse transcriptase (Agilent Technologies, Santa Clara, CA, US) was amplified with Q5 polymerase (New England BioLabs, US) and gene-specific primers. The 5′ and 3’ ends of the genome were obtained using a Rapid Amplification of cDNA Ends (RACE) kit (Life Technologies, Inc., Carlsbad, CA, US). The sequences were assembled with Sequencher DNA sequence analysis software version 2.1 (Gene Codes, Ann Arbor, MI, US) and the completed genome sequence, designated as SARS-CoV-2 UF-30, submitted to GenBank.

Nannu Shankar S., Witanachchi C.T., Morea A.F., Lednicky J.A., Loeb J.C., Alam M.M., Fan Z.H., Eiguren-Fernandez A, & Wu C.Y. (2021). SARS-CoV-2 in residential rooms of two self-isolating persons with COVID-19. Journal of Aerosol Science, 159, 105870.

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Comprehensive HIV-1 Genome Sequencing

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Plasma viral RNA was purified from pelleted virus particles by centrifuging one milliliter of plasma at 20,000g × 60 min at 4 °C, removing 860 µl of cell-free supernatant and resuspending the pellet in the remaining 140 µl, to finally extract viral RNA using QIAamp Viral RNA Mini kit (Qiagen; Valencia, CA). Viral RNA was reverse-transcribed using AccuScript High Fidelity Reverse Transcriptase (Stratagene Agilent; Santa Clara, CA) and five previously described antisense external primers (Pan-HIV-1-1R [68 (link)], 1R, 2R, 3R, and 4R [69 (link)]) in 20 µl reaction mixtures containing 1 mM dNTPs, 10 mM DTT and 10 units of RNAse inhibitor. Six overlapping fragments, covering almost the entire HIV-1 genome, were amplified using a series of external and nested primers and Phusion High-Fidelity DNA Polymerase (NEB, Ipswich, MA) with defined cycling conditions as previously described [68 (link), 69 (link)], i.e., 5′LTR (675 bp; HXB2 coordinates 120 to 794), 5′LTR-gag/p7 (1269 bp; 658 to 1924), gag/p24-pol/RT (2327 bp; 1137 to 3463), pol/RT-vif (2259 bp; 2976 to 5234), pol/int-env/gp120 (2921 bp; 4602 to 7522), and env/gp120-3′LTR (2587 bp; 6858 to 9444).

Weber J., Gibson R.M., Sácká L., Strunin D., Hodek J., Weberová J., Pávová M., Alouani D.J., Asaad R., Rodriguez B., Lederman M.M, & Quiñones-Mateu M.E. (2017). Impaired human immunodeficiency virus type 1 replicative fitness in atypical viremic non-progressor individuals. AIDS Research and Therapy, 14, 15.

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