Maxima h minus reverse transcriptase kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Maxima H Minus Reverse Transcriptase Kit is a laboratory reagent used for the reverse transcription of RNA into complementary DNA (cDNA). The kit contains the Maxima H Minus Reverse Transcriptase enzyme, which is designed to efficiently convert RNA into cDNA for various downstream applications, such as real-time PCR and gene expression analysis.

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24 protocols using maxima h minus reverse transcriptase kit

Quantitative PCR of Immune Genes in CD4+ T Cells

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Total RNA was isolated from CD4⁺ T cells with TRIzol reagent (Invitrogen) as described in the manufacturer’s instructions and reverse-transcribed using the Maxima H Minus Reverse Transcriptase Kit (Thermo Fisher Scientific, no. EP0753). All qPCRs were run on Bio-Rad CFX96 real-time system using iTaq Universal SYBR Green Supermix (Bio-Rad, no. 1725124), and β-actin was used as an internal control to normalize the data across different samples.
Primer sequences used for qPCR are as follows: Actb (forward, 5′-AGTGTGACGTTGACATCCGT-3′; reverse, 5′-GCAGCTCAGTAACAGTCCGC-3′), Alkbh5 (forward, 5′-CGCGGTCATCAACGACTACC-3′; reverse, 5′- ATGGGCTTGAACTGGAACTTG-3′), Fto (forward, 5′-GCCTCGGTTTAGTTCCACTCAC-3′; reverse, 5′-GTCGCCATCGTCTGAGTCATTG-3′), Ifng (forward, 5′-CAGCAACAGCAAGGCGAAA-3′; reverse, 5′-CTGGACCTGTGGGTTGTTGAC-3′), Cxcl2 (forward, 5′-CCCTGCCAAGGGTTGACTTC-3′; reverse, 5′-GCAAACTTTTTGACCGCCCT-3′), Cxcl10 (forward, 5′-CCTATGGCCCTCATTCTCAC-3′; reverse, 5′-CTCATCCTGCTGGGTCTGAG-3′), and Myc peak (forward, 5′-GCTTCGAAACTCTGGTGCAT-3′; reverse, 5′-AATTCCAGCGCATCAGTTCT-3′).

Zhou J., Zhang X., Hu J., Qu R., Yu Z., Xu H., Chen H., Yan L., Ding C., Zou Q., Ye Y., Wang Z., Flavell R.A, & Li H.B. (2021). m6A demethylase ALKBH5 controls CD4+ T cell pathogenicity and promotes autoimmunity. Science Advances, 7(25), eabg0470.

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RNA Isolation and cDNA Synthesis

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All kits used in this study were used following the instructions of the manufacturer. Total RNA was isolated using the Bio & Sell RNA mini Kit (Bio&Sell e.K., Feucht, Germany). To evaluate the abundance of SPO11 transcripts in generative tissue, fresh young flowers were used for RNA isolation. In the case of C. papaya, flowers were stored in RNAshield (Zymo research Europe GmbH, Freiburg, Germany) prior to RNA isolation. To check the abundance in vegetative tissue, leaf material was used. In the case of C. papaya no leaf material was available so fruit exocarp tissue was utilized instead. To check expression in P. patens 6-week old gametophores were used for RNA Isolation. Isolated RNA was treated with DNase I (Thermo Fisher Scientific, Germany). To check contamination with genomic DNA in the treated RNA, a PCR was performed with RNA as a template. No contamination was found in the RNA samples after DNase treatment (data not shown). cDNA was produced using an anchored oligo dT Primer with the Maxima H Minus Reverse Transcriptase Kit (Thermo Fisher Scientific, Germany) using 2–4 μg of total RNA as a template for the RT-reaction.

Sprink T, & Hartung F. (2014). The splicing fate of plant SPO11 genes. Frontiers in Plant Science, 5, 214.

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Droplet Digital PCR Quantification of EWS Rearrangements

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RNA was reverse-transcribed to cDNA with a Maxima H Minus Reverse Transcriptase Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. The reverse transcription reaction was performed at 55 °C for 30 min and 85 °C for 5 min. Thereafter, cDNA was detected with ddPCR Supermix for Probes (No dUTP, Bio-Rad). EWS rearrangements were detected using self-designed primers and probes (Table S1, Supporting Information). SRY transcript was detected using a commercial primer/probe kit (Catalog# 4331182; Assay ID: Hs00976796_s1, Thermo Fisher Scientific). Droplets containing ddPCR reaction were transferred into a 96-well plate and sealed. ddPCR reaction was performed at 96 °C for 10 min, followed by 40 cycles (94 °C for 30 s and 60 °C for 60 s) and 98 °C for 10 min. The DNA amplicons contained in droplets were detected by a QX200 Droplet Reader in combination with a QuantaSoft software package.

Dong J., Zhang R.Y., Sun N., Hu J., Smalley M.D., Zhou A., Yue H., Rothermich W., Chen M., Chen J., Ye J., Teng P.C., Qi D., Toretsky J.A., Tomlinson J.S., Li M., Weiss P.S., Jonas S.J., Federman N., Wu L., Zhao M., Tseng H.R, & Zhu Y. (2020). Coupling Nanostructured Microchips with Covalent Chemistry Enables Purification of Sarcoma-Derived Extracellular Vesicles for Downstream Functional Studies. Advanced functional materials, 30(49), 2003237.

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RNA Isolation and qPCR from BMDMs

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BMDMs were plated at 1–2×10⁶ cells per well on a flat bottom non-adherent 12-well plates. Following treatment, cells were washed once with PBS, and gently dislodged from the plate with PBS. Cells were pelleted and then processed using Qiagen RNeasy Mini Plus Kit (Qiagen) using the manufacturer’s protocol. RNA quantity was assayed using a NanoDrop Microvolume Spectrophotometer (Thermo). RNA was sent for RNA-sequencing or converted to cDNA using Maxima H Minus Reverse Transcriptase Kit and using the manufacturer’s protocol (Thermo). qPCR was conducted using Sigma KiCqStart predesigned SYBR green primers and iTaq™ Universal SYBR® Green Supermix (Bio-Rad). Relative mRNA amounts were compared to Gapdh mRNA levels. mRNA for RNAseq analysis was purified using PolyA⁺ selection and processed by the Yale Center for Genome Analysis using standard methodology and sequenced on a HiSeq2000 with 75bp pair-ended reads.

Solis A.G., Bielecki P., Steach H.R., Sharma L., Harman C.C., Yun S., Palm N.W., de Zoete M.R., Warnock J.N., To S.D., York A.G., Mack M., Schwartz M.A., Cruz C.S., Jackson R, & Flavell R.A. (2019). Mechanosensation of Cyclical Force by PIEZO1 is Essential for Innate Immunity. Nature, 573(7772), 69-74.

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Quantitative Gene Expression Analysis

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We isolated total RNA using the RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNA digestion according to the manufacturer’s recommendations. We measured the concentration of isolated RNA with Implen NanoPhotometerTM (Implen, Munich, Germany). 1 µg of total RNA was reverse-transcribed into cDNA using a Maxima H minus Reverse Transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA). We used the equivalent of 20 ng for RT-qPCR on ABI-PRISM 7500. Each reaction was run in triplicates using TaqMan® gene expression assays (Hprt1: Rn01527840_m1; INS1: Rn02121433_g1; INS2: Rn01774648_g1 and GC: Rn00561256) andTaqMan® Fast Advanced Master Mix (Thermofisher Scientific, USA). We normalized gene expression of target genes to the expression of Hprt1. We calculated the fold change of expression using the ΔΔCt method.

Pavlíková N., Daniel P., Šrámek J., Jelínek M., Šrámková V., Němcová V., Balušíková K., Halada P, & Kovář J. (2019). Upregulation of vitamin D-binding protein is associated with changes in insulin production in pancreatic beta-cells exposed to p,p′-DDT and p,p′-DDE. Scientific Reports, 9, 18026.

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Quantitative RT-PCR analysis of RNA

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Four hundred ng of total RNA isolated from blood samples was used for cDNA synthesis using random hexamers and the Maxima H Minus Reverse Transcriptase Kit according to the manufacturer’s protocol (Thermo Fisher Scientific, Carlsbad, USA). RT-PCR was performed using 2 μL cDNA, a forward primer located in exon 11 and a reverse primer located in exon 17 (see Supplementary Material, Table S1). For quantitative analysis, RT-PCR was performed using the same primers, but the reverse primer was 5′ FAM (6-carboxyfluorescein) labeled.
FAM-labeled RT-PCR products were mixed with 0.5 μL of GeneScan ROX500 size standard (Life Technologies, Darmstadt, Germany) and 8.5 μL of Hi-Di Formamide (Life Technologies) in a total volume of 10 μL. Mixes were separated by capillary electrophoresis on an ABI 3130XL Genetic Analyzer instrument (Life Technologies). The area-under-the-curve (AUC) was calculated with GeneMapper 5 software (Life Technologies). Ratios of RT-PCR products were determined as the AUC for individual peaks divided by the sum of AUC of all peaks.
Subcloning of RT-PCR products was performed using the NEB® PCR Cloning Kit (New England Biolabs, Frankfurt, Germany) according to manufacturer’s instructions.

Weisschuh N., Marino V., Schäferhoff K., Richter P., Park J., Haack T.B, & Dell’Orco D. (2021). Mutations at a split codon in the GTPase-encoding domain of OPA1 cause dominant optic atrophy through different molecular mechanisms. Human Molecular Genetics, 31(5), 761-774.

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cDNA Synthesis from T. leucotreta RNA

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The RNA of
T. leucotreta, isolated using the four methods described earlier, was converted to cDNA using the Maxima H Minus Reverse Transcriptase kit with a random hexamer primer (5′-d(NNNNNN) -3 (Thermo Scientific, Wilmington, DL). The sample was incubated at 25°C for 10 min followed by 30 min at 50°C. The reaction was terminated by heating the sample to 85°C for 5 min. cDNA was then tested in a PCR using the conditions described earlier to determine its applicability for a basic downstream application.

Ridgeway J.A, & Timm A.E. (2014). Comparison of RNA Isolation Methods From Insect Larvae. Journal of Insect Science, 14, 268.

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RNA Extraction and RT-PCR for AsMaV Detection

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A minimum of five symptomatic leaves from the same tree were selected and neatly piled up. Then, approximately 1–2 cm² of the leaves (0.2–0.3 g) were used for RNA extraction. Total RNA was isolated according to the protocol described by Boom et al. [46 (link)] and 1.5 µg of the extracted RNA was applied as a template for cDNAs synthesis using random hexamers and Maxima H Minus Reverse Transcriptase kit (Thermo Scientific™, Waltham, MA, USA) following the protocol provided by the manufacturer. The presence of AsMaV in the collected samples was confirmed through a series of RT–PCRs employing specific primers (Supplementary Table S2). These primers were designed to hybridize with AsMaV-RNA3 to RNA5, following established method described elsewhere [5 (link),47 (link)]. To design the novel primers, we utilized the RNA1 to RNA5 sequences of AsMaV isolates E55089 (GenBank accession No. LR74246, LR742462–65) as a reference. The detailed characteristics of these newly designed primers are provided in Supplementary Table S2. Notably, the isolate E55089 and its corresponding genomic molecules were used as a standard for nucleotide and amino acid positioning.

Nourinejhad Zarghani S., Al Kubrusli R., Iancev S., Jalkanen R., Büttner C, & von Bargen S. (2023). Molecular Population Genetics of Aspen Mosaic-Associated Virus in Finland and Sweden. Viruses, 15(8), 1678.

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Quantifying ALK and ROS1 Rearrangements

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The CTCs released from Click Chips were collected in a 1.5-ml RNase-free Eppendorf tube and lysed by TRI Reagent [1:3, (v/v), Zymo Research Corp.]. The collected RNA was purified using a Direct-zol RNA MicroPrep Kit (Zymo Research Corp.) according to the manufacturer’s protocol. The purified RNA was reverse-transcribed to cDNA using a Thermo Scientific Maxima H Minus Reverse Transcriptase Kit according to the manufacturer’s instructions. Samples of cDNA were detected with a PrimePCR ddPCR Expert Design Assay Kit from Bio-Rad that covers 26 ALK gene rearrangement subtypes and 14 ROS1 gene rearrangement subtypes. Data were analyzed using the QuantaSoft software package to calculate the corresponding copy numbers of ALK or ROS1 rearrangements detected from individual samples.

Dong J., Jan Y.J., Cheng J., Zhang R.Y., Meng M., Smalley M., Chen P.J., Tang X., Tseng P., Bao L., Huang T.Y., Zhou D., Liu Y., Chai X., Zhang H., Zhou A., Agopian V.G., Posadas E.M., Shyue J.J., Jonas S.J., Weiss P.S., Li M., Zheng G., Yu H.H., Zhao M., Tseng H.R, & Zhu Y. (2019). Covalent chemistry on nanostructured substrates enables noninvasive quantification of gene rearrangements in circulating tumor cells. Science Advances, 5(7), eaav9186.

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EV-Derived mRNA Analysis via ddPCR

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EV-derived mRNA was reverse-transcribed to cDNA using a Maxima H Minus Reverse Transcriptase Kit (Thermo Fisher Scientific). The EV-derived mRNA was added into a reaction solution containing 1× RT Buffer, dNTPs (0.5 mM), Random Hexamer (8 μM), Maxima H Minus Reverse Transcriptase (6.5 U μL⁻¹), and RNase inhibitor (1 U μL⁻¹). The reaction was run at 55 °C for 30 min and then 85 °C for 5 min. The cDNA generated from EV-derived mRNA was detected by the PrimePCR ddPCR Expert Design Assay Kit (dHsaEXD73338942, ROS1 rearrangements) or PrimePCR ddPCR Mutation Assay Kit (dHsaCP2000020, EGFR T790M mutation, Bio-Rad, USA) according to the manufacturer’s instructions. For ddPCR, droplets were generated within a DG8 Cartridge which was preloaded with sample (20 μL) and droplet generation oil (70 μL) for each sample. All droplets were transferred into a 96-well plate accordingly and sealed with a PX1 PCR Plate Sealer. A programmed Thermal Cycler was set at 96 °C for 10 min, followed by 40 cycles of 94 °C for 30 s and 60 °C for 60 s, and finally 98 °C for 10 min. The droplets containing amplicons were quantified with a QX200 Droplet Reader using the QuantaSoft software package.

Dong J., Zhang R.Y., Sun N., Smalley M., Wu Z., Zhou A., Chou S.J., Jan Y.J., Yang P., Bao L., Qi D., Tang X., Tseng P., Hua Y., Xu D., Kao R., Meng M., Zheng X., Liu Y., Vagner T., Chai X., Zhou D., Li M., Chiou S.H., Zheng G., Di Vizio D., Agopian V.G., Posadas E., Jonas S.J., Ju S.P., Weiss P.S., Zhao M., Tseng H.R, & Zhu Y. (2019). Bio-Inspired NanoVilli Chips for Enhanced Capture of Tumor-Derived Extracellular Vesicles: Toward Non-Invasive Detection of Gene Alterations in Non-Small Cell Lung Cancer. ACS applied materials & interfaces, 11(15), 13973-13983.

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