Maxima reverse transcriptase

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, China, Lithuania, Italy, United Kingdom, France

Maxima Reverse Transcriptase is a thermostable and highly sensitive enzyme used for the conversion of RNA into cDNA. It is designed for use in reverse transcription reactions.

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Maxima H Minus M-MuLV reverse transcriptase Maxima H Reverse Transcriptase Thermo Scientific Maxima H Minus Reverse Transcriptase Kit Maxima Reverse Transcriptase kit Maxima Reverse H minus Reverse Transcriptase Maxima reverse transcriptase enzyme Maxima reverse transcriptase mix Maxima Reverse Transcriptase system Maxima H Minus Reverse Transcriptase Kit Maxima RT reverse transcriptase

330 protocols using maxima reverse transcriptase

Confirming SARS-CoV-2 in Pouched Lamprey

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To further confirm the presence of the novel coronavirus in the pouched lamprey, total RNA was re-extracted from the 12 HiSeq-sequenced samples using the methods described above. The total RNA was then reverse transcribed (RT) using Maxima Reverse Transcriptase kit (Thermo Scientific) and ReadyMade Random Hexamers (Integrated DNA Technologies). The Maxima Reverse Transcriptase (Thermo Scientific) protocol was followed and did not include the GC-rich template options: 1 μl template total RNA, 1 μl ReadyMade Random Hexamers (10 mM), 1 μl dNTP mix (10 mM), 11.5 μl nuclease-free water, 4 μl 5X RT Buffer, 0.5 μl (20U) RiboLock RNase Inhibitor (Thermo Scientific), and 1 μl Maxima Reverse Transcriptase. The final volume (20 μl) was stored at −20°C until used in PCRs. See Supplementary Table S1 for the primer set used.

Miller A.K., Mifsud J.C., Costa V.A., Grimwood R.M., Kitson J., Baker C., Brosnahan C.L., Pande A., Holmes E.C., Gemmell N.J, & Geoghegan J.L. (2021). Slippery when wet: cross-species transmission of divergent coronaviruses in bony and jawless fish and the evolutionary history of the Coronaviridae. Virus Evolution, 7(2), veab050.

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Quantifying Nrf2 and Nqo1 Expression

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To evaluate mRNA expression levels of Nrf2 and its target gene Nqo1, the Hippocampus and Cortex of mice between 8 and 12 weeks old were isolated and collected in RNAlater (Invitrogen). RNA extraction was performed using TRIzol (Invitrogen) and 500 ng RNA was synthesized to cDNA using the Maxima Reverse Transcriptase (Thermo Fisher).
For each quantitative real-time PCR (qPCR) reaction 40 ng cDNA was combined with Maxima Probe Rox qPCR Mastermix (Thermo Fisher) supplemented with adequate Taqman primers (Thermo Fisher) and 18S rRNA (Thermo Fisher) as an endogenous control using the StepOnePlus Real-Time PCR System (Applied Biosystems). Analysis was conducted with StepOne software v2.1. The following Taqman primers were used: Nrf2 (Mm00477784_m1) and Nqo1 (Mm01253561_m1).
Data were analyzed using the ∆∆CT method followed by relative quantification (2^−∆∆CT).

Gola L., Bierhansl L., Csatári J., Schroeter C.B., Korn L., Narayanan V., Cerina M., Abdolahi S., Speicher A., Hermann A.M., König S., Dinkova-Kostova A.T., Shekh-Ahmad T., Meuth S.G., Wiendl H., Gorji A., Pawlowski M, & Kovac S. (2023). NOX4-derived ROS are neuroprotective by balancing intracellular calcium stores. Cellular and Molecular Life Sciences, 80(5), 127.

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Quantifying Transgene Expression in Mice

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To determine transgene copy number, genomic DNA was isolated and purified from lysed toe tissue and primed for genomic Jpx and Xist genes. Presence of the transgene was also confirmed by priming to the BAC8 vector [36 (link)]. Transgene copy number was determined by normalizing the genomic Jpx and Xist copy numbers to the X-linked Hprt gene (internal control) and comparing to the wildtype male or female samples. To measure RNA expression, cultured E13.5 mEFs or minced embryo tissue (E7.5/ 8.5) were homogenized in TRIzol Reagent (Invitrogen); chloroform and isopropanol were used to extract and precipitate RNA; and the RNA was treated with TURBO DNaseI (Life Technology) before reverse transcription with Maxima Reverse Transcriptase (Thermo Fisher). qRT-PCR was then performed on a BioRad CFX96 Real-Time PCR system. Jpx and Xist primers targeted mature transcripts as described in the Supplemental Experimental Procedures. Gapdh expression was used as an internal control [38 (link)].

Carmona S., Lin B., Chou T., Arroyo K, & Sun S. (2018). LncRNA Jpx induces Xist expression in mice using both trans and cis mechanisms. PLoS Genetics, 14(5), e1007378.

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Analyzing OGT Expression by qPCR

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Total RNA was purified[17 (link)] and transcribed into cDNA using Maxima reverse transcriptase (Thermo Scientific). GAPDH and OGT mRNA was detected by real time qPCR using iTaq™ Universal Probes Supermix (Bio-Rad) and TaqMan Gene Expression Assay (Thermo Scientific). Relative OGT/GAPDH gene expression was calculated by the ΔΔCt method[21 (link)].

Herzog K., Bandiera S., Pernot S., Fauvelle C., Jühling F., Weiss A., Bull A., Durand S.C., Chane-Woon-Ming B., Pfeffer S., Mercey M., Lerat H., Meunier J.C., Raffelsberger W., Brino L., Baumert T.F, & Zeisel M.B. (2019). A functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity. Gut, 69(2), 380-392.

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Quantitative RT-PCR Analysis of Helicobacter pylori

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RNA was extracted from three independent sets of H. pylori cultures in the logarithmic phase of growth (OD₆₀₀ ~ 0.4–0.7). RNA was extracted using a Total RNA Extraction Plus kit (A&A Biotechnology) according to the manufacturer’s protocol, and further treated with RNase-free DNAseI (Thermo Scientific). Reverse transcription (RT) reactions were carried out on 0.5 μg of total RNA in 20 μl using Maxima Reverse Transcriptase (Thermo Scientific) and random hexamer primers in the presence of RiboLock RNase Inhibitor (Thermo Scientific), as described by the manufacturer. mRNA levels of the selected H. pylori genes were quantified by qPCR, performed on a CFX96 Touch Real-Time PCR Detection System (BioRad) using SensiFAST SYBR No-ROX (BioLine) and the following parameters: 96 °C for 2 min, followed by 40 three-step amplification cycles consisting of 5 s at 96 °C, 10 s at 60 °C and 10 s at 72 °C. Reaction mixtures (15 µl) contained qPCR mix (7.5 μl), cDNA (1 μl of 50x diluted RT reaction) and primers (0.3 µM each). The following primer pairs were used: secA, H28-H29; nifS, H30-H31 and 16SrRNA, H32-H33. The relative quantity of mRNA for each gene was determined by reference to the mRNA levels of H. pylori 16SrRNA.

Zawilak-Pawlik A., Zarzecka U., Żyła-Uklejewicz D., Lach J., Strapagiel D., Tegtmeyer N., Böhm M., Backert S, & Skorko-Glonek J. (2019). Establishment of serine protease htrA mutants in Helicobacter pylori is associated with secA mutations. Scientific Reports, 9, 11794.

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RNA Isolation and qRT-PCR Analysis

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RNA was isolated using innuPREP RNA Mini Kit (Jena Analytik, Jena, Germany) according to the manufacturer’s protocol. For reverse transcription 1 μg of RNA per sample was used. The reaction was performed with Maxima Reverse Transcriptase using Oligo(dT)₁₈ primer (both Thermo Fisher Scientific, Munich, Germany). Prior to qRT-PCR, template cDNA was diluted 1:40 in ddH₂O, mixed with primers (2 μM), as well as SyBRGreen Master Mix (Applied Biosystems by Thermo Fisher Scientific, Munich, Germany) according to the manufacturer’s protocol. The PCR was performed under standard conditions in a MX3000P sequence detection system (Stratagene by Agilent, Waldbronn, Germany).

Dollt C., Michel J., Kloss L., Melchers S., Schledzewski K., Becker K., Sauer A., Krewer A., Koll F, & Schmieder A. (2018). The novel immunoglobulin super family receptor SLAMF9 identified in TAM of murine and human melanoma influences pro-inflammatory cytokine secretion and migration. Cell Death & Disease, 9(10), 939.

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Single-cell Transcriptomics of Organoid Cultures

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At d100, cell lines used were 1013 and 1581 for WT and L75Pfs clones 9 and 11. D170 organoids were 1013 for WT and clones 7 and 9 for L75Pfs. Organoids were dissociated to single cells with papain (Worthington) to 1 mg/ml and 5 μL DNase (Roche) per mL, using 200 uL papain mix per organoid. After 1–2 h when organoids appeared fully dissociated the reaction was quenched with media containing 10% FBS (Gibco). Single cells were resuspended in HBSS (Gibco) and 0.1 mg/mL BSA at 120,000 cells per mL. Single-cell capture was performed using a home-made Dropseq setup according to the published Dropseq protocol^{21 (link)}. Cells were combined in oil (Biorad) and barcoded beads (Chemgenes) in ~1 nL droplets. Droplets were broken using 6× SSC and perfluorooctanol (Sigma) to collect beads. Reverse transcription was performed using Maxima reverse transcriptase (Thermo Fisher Scientific) and cDNA was amplified using Kapa (Roche). cDNA was quantified using a Bioanalyzer DNA High Sensitivity Chip (Agilent). cDNA was fragmented and libraries were created using the Nextera XT library prep kit (Illumina). Libraries with quantified by Qubit dsDNA HS (Thermo Fisher Scientific) and sequenced via Illumina HiSeq 2500.

Kallman A., Capowski E.E., Wang J., Kaushik A.M., Jansen A.D., Edwards K.L., Chen L., Berlinicke C.A., Joseph Phillips M., Pierce E.A., Qian J., Wang T.H., Gamm D.M, & Zack D.J. (2020). Investigating cone photoreceptor development using patient-derived NRL null retinal organoids. Communications Biology, 3, 82.

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High-Throughput Antibody Repertoire Sequencing

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Antibody repertoire sequencing was performed as previously described (23 (link), 29 (link)). Briefly, plasmablasts were sorted into 96-well plates containing lysis buffer (10mM Tris with 1U/μL RiboLock RNAse inhibitor) and stored at −80C. One of 96 unique well-specific DNA barcodes was added to the cDNA of each cell by template switching during reverse transcription with Maxima Reverse Transcriptase (Thermo Fisher Scientific). Well ID-tagged cDNA was pooled from each plate, and plate-specific indices were added during PCR with primers specific for the HC and LC constant regions. Gamma, Kappa, and Lambda primers were as described in (23 (link)), and the nested Alpha gene-specific primer sequences were 1: ATT CGT GTA GTG CTT CAC GTG; 2: CTA TGC GCC TTG CCA GCC CGC GGG AAG ACC TTG GGG CTG GT. Barcoded amplicons from multiple plates were pooled prior to the addition of sequencing adaptors and final purification with AMPure XP beads (Beckman Coulter). Samples sequenced prior to Fall 2014 underwent Roche 454 sequencing using Lib-L adaptors and Titanium chemistry. Following the development of 600 bp read lengths on the Illumina MiSeq platform, samples underwent 2 × 300 MiSeq analysis.

Kinslow J.D., Blum L.K., Deane K.D., Demoruelle M.K., Okamoto Y., Parish M., Kongpachith S., Lahey L.J., Norris J.M., Robinson W.H, & Holers V.M. (2016). IgA Plasmablasts are Elevated in Subjects At Risk for Future Rheumatoid Arthritis. Arthritis & rheumatology (Hoboken, N.J.), 68(10), 2372-2383.

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Isolation and Analysis of RNA

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Con A was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China), batch no. C8110. Chloral hydrate and UNlQ-10 Column Total RNA Isolation Kit were gained from Sangon Biotech Co., Ltd. (Shanghai, China), batch number: A600288 and B511321. Maxima Reverse Transcriptase was gotten from Thermo Fisher Scientific (China) Co., Ltd. (Shanghai, China), batch no. EP0743.

Liu Y., Chen H., Hao J., Li Z., Hou T, & Hao H. (2020). Characterization and functional prediction of the microRNAs differentially expressed in a mouse model of concanavalin A-induced autoimmune hepatitis. International Journal of Medical Sciences, 17(15), 2312-2327.

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DNA and RNA Extraction from Whole Blood

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DNA was extracted from whole blood using a QIAamp DNA Blood Mini Kit (Qiagen, Venlo, The Netherlands) in accordance with the manufacturer’s instructions. DNA purity and concentration were assessed using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
Total RNA was isolated from blood collected in a PAXgene Blood RNA Tube IVD (Qiagen) and purified using a PAXgene Blood RNA Kit (Qiagen). RNA samples (1 μg) were reverse transcribed using Maxima reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA).

Stella A., Lastella P., Loconte D.C., Bukvic N., Varvara D., Patruno M., Bagnulo R., Lovaglio R., Bartolomeo N., Serio G, & Resta N. (2018). Accurate Classification of NF1 Gene Variants in 84 Italian Patients with Neurofibromatosis Type 1. Genes, 9(4), 216.

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