Mint 2 cdna synthesis kit

Manufactured by Evrogen

The Mint-2 cDNA synthesis kit is a tool used for the reverse transcription of RNA into complementary DNA (cDNA). It provides the necessary components, including enzymes and buffers, to facilitate the conversion of RNA templates into single-stranded cDNA molecules.

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Mint kit (11 citations) Mint cDNA synthesis kit (9 citations) MINT cDNA kit (2 citations) Mint-2 (2 citations)

11 protocols using mint 2 cdna synthesis kit

Quantifying Transgene Expression via RT-qPCR

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Total RNA was isolated using the TRIzol method (Gibco/Life Technologies) or RNeasy kits (Qiagen). Three micrograms of total RNA isolated from transiently transfected cells was reverse transcribed using the MMLV RT kit (Evrogen) with an oligo(dT)₂₀ primer, according to the manufacturer's protocol. The Mint-2 cDNA synthesis kit (Evrogen) was used for total cDNA amplification.
Real-time PCR (Stratagene MX3005, qPCRmix-HS SYBR+ROX Kit, Evrogen) was used to quantify the levels of target transcripts. The abundance of the TagGFP2- and Katushka-encoding transcripts was measured with the primers 5′-cgccatcagcaaggaccg and 5′-actggtggggtcaattctttgc for TagGFP2 and 5′-tgagagcggattgacaggccat and 5′-gagtccggattgatcccccagtttgct for Katushka (0.2 μM each). In each sample, the level of TagGFP2-encoding transcript was normalized to the level of Katushka-encoding transcript. The primers 5′-cgctaccggactccag and 5′-caagttaacaacaacaattgc were used to control for the presence of non-spliced or alternatively spliced forms of the TagGFP2-encoding transcript.

Pereverzev A.P., Gurskaya N.G., Ermakova G.V., Kudryavtseva E.I., Markina N.M., Kotlobay A.A., Lukyanov S.A., Zaraisky A.G, & Lukyanov K.A. (2015). Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Scientific Reports, 5, 7729.

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cDNA Synthesis and Normalization

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Full-length-enriched double-stranded cDNA was then synthesized using the Mint-2 cDNA synthesis kit (Evrogen, Moscow, Russia/ Cat # SK005). To reduce the prevalence of abundant transcripts, the resulting double-stranded cDNAs were normalized using the Evrogen Trimmer-2 cDNA normalization kit (Evrogen, Moscow, Russia/ Cat # NK003) [26 (link)]. The resulting normalized cDNA midgut library was then submitted to 454- high-throughput pyrosequencing.

Valencia A., Wang H., Soto A., Aristizabal M., Arboleda J.W., Eyun S.I., Noriega D.D, & Siegfried B. (2016). Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts. PLoS ONE, 11(3), e0151001.

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Transcriptome Analysis of Powdery Mildew Fungus

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Total RNA was isolated from epiphytic mycelium and conidia of P. xanthii collected from two different heavily powdery mildew-infected zucchini cotyledons by carefully removing the epiphytic fungal biomass with a spatula, immediately frozen in liquid nitrogen, and stored at -80°C until use. Total RNA was extracted using TRI Reagent (Sigma-Aldrich, Saint Louis, MO) and NucleoSpin RNA Plant (Macherey-Nagel, Düren, Germany) according to the manufacturer´s instructions. Total RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). RNA quality and quantity were measured by running 1 μl of sample on an Agilent Bioanalyzer 2100 using a RNA Pico 600 chip (Agilent Technologies, Santa Barbara, CA). A non-normalised cDNA library was synthesised from 1.5 μg total RNA with the Mint-2 cDNA synthesis kit (Evrogen, Moscow, Russia). The 454 libraries obtained in this manner were immobilised on beads and clonally amplified using the GS FLX Titanium LV emPCR kit (454 Life Sciences, Branford, CT). The libraries were then sequenced using the GS FLX Titanium Sequencing Kit XLR70 (454 Life Sciences) and GS FLX Titanium PicoTiterPlate kit on a GS FLX instrument (454 Life Sciences). All kits were used according to the manufacturer’s instructions.

Vela-Corcía D., Bautista R., de Vicente A., Spanu P.D, & Pérez-García A. (2016). De novo Analysis of the Epiphytic Transcriptome of the Cucurbit Powdery Mildew Fungus Podosphaera xanthii and Identification of Candidate Secreted Effector Proteins. PLoS ONE, 11(10), e0163379.

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Transcriptome Profiling of Plant Samples

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In total, 50 samples (10 plants on five sampling days) were used for RNA extraction. Total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Extracted RNA was sent to Chronix Biomedical GmbH (Göttingen, Germany) for library preparation and sequencing. RNA quality and integrity were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA library preparations were conducted using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs, Frankfurt am Main, Germany). Additionally, a normalized composite sample was created to enhance the de novo transcriptome assembly. For that, extracted RNA of all 50 samples was pooled to a single composite sample. The cDNA library was prepared using the Mint-2 cDNA synthesis kit (Evrogen, Moscow, Russia), and normalized using the Trimmer-2 cDNA normalization kit (Evrogen, Moscow, Russia). The 51 libraries (50 single samples from the five time points plus one composite sample) were paired-end sequenced on five lanes on an Illumina HiSeq2000 platform (Illumina, San Diego, CA, USA). Each library was uniquely tagged with a barcode to allow library pooling for sequencing, and control and treatment samples were always sequenced together on one lane.

Müller M., Seifert S., Lübbe T., Leuschner C, & Finkeldey R. (2017). De novo transcriptome assembly and analysis of differential gene expression in response to drought in European beech. PLoS ONE, 12(9), e0184167.

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Cloning and Sequencing of Vanilla and Glechoma cDNAs

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A complementary DNA (cDNA) library made from a 6-month-old V. planifolia pod was kindly provided by Evolva A/S Denmark. The cDNA library was inserted in a pYES2 vector (Invitrogen) ( http://tools.invitrogen.com/content/sfs/manuals/pyes2_man.pdf). cDNA from Glechoma hederacea was made from material sourced in Basel, Switzerland. Total RNA was isolated using the RNeasy plant kit (Qiagen) and cDNA was made using the Mint2 cDNA synthesis kit (Evrogen) ( www.evrogen.com).
Candidate genes identified from the transcriptome data were amplified from the cDNA library by PCR with gene-specific primers (Supplementary Tables 1 and 3) to obtain full-length sequences. The PCR products were subsequently cloned in blunt-II-topo vector (Invitrogen) in E. coli. Plasmids were purified using the miniprep kit (Qiagen) and gene sequences were confirmed by sequencing.

Gallage N.J., Hansen E.H., Kannangara R., Olsen C.E., Motawia M.S., Jørgensen K., Holme I., Hebelstrup K., Grisoni M, & Møller B.L. (2014). Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme. Nature Communications, 5, 4037.

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Tentacle Transcriptome Sequencing

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Tentacle treatment. Tentacles were cut off using scissors, frozen in liquid nitrogen, and stored at –70 °C up to RNA isolation. Total RNA was extracted with TRIzol Reagent (Life Technologies, US). The yield and purity were assessed using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, US), with the RNA integrity determined by the RNA integrity number using a Bioanalyser 2100 (Agilent Technologies, US). The PCR-based cDNA library was created following the instructions for the Mint-2 cDNA synthesis kit (Evrogen, Russia). Adapters PlugOligo-3M and CDS-4M were used for cDNA synthesis.
Ion torrent sequencing. For library preparation, amplified cDNAs (100 ng of each sample) were fragmented by 400–500 bp using the Covaris S220 System (Covaris, Woburn, Massachusetts, US). Next, the Ion Xpress Plus Fragment Library Kit (Life Technologies) was employed for barcode shotgun-library sample preparation. To conduct emulsion PCR, the Ion PGM Template OT2 400 Kit (Life Technologies) was utilised. Sequencing was performed in accordance with manufacturer protocol for the genome analyser Ion Torrent PGM (Life Technologies), using an Ion 318 chip and Ion PGM Sequencing 400 Kit v2 (Life Technologies).

Babenko V.V., Mikov A.N., Manuvera V.A., Anikanov N.A., Kovalchuk S.I., Andreev Y.A., Logashina Y.A., Kornilov D.A., Manolov A.I., Sanamyan N.P., Sanamyan K.E., Kostryukova E.S., Kozlov S.A., Grishin E.V., Govorun V.M, & Lazarev V.N. (2017). Identification of unusual peptides with new Cys frameworks in the venom of the cold-water sea anemone Cnidopus japonicus. Scientific Reports, 7, 14534.

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Tissue-specific transcriptome profiling

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Total RNA was extracted from the tissue fragments isolated by laser microdissection using an ExtractRNA Kit (Evrogen, Russia) (for details, see Supplementary Methods). Tissue-specific cDNA libraries were created using the Mint-2 cDNA Synthesis Kit (Evrogen, Russia) according to the manufacturer’s instructions. The adapters PlugOligo-3 M and CDS-4 M were used for cDNA synthesis. The normalization of cDNA was performed using the DNS (duplex-specific nuclease) in accordance with the manufacturer’s protocol (Evrogen, Russia). Normalized and non-normalized cDNA libraries (100 ng of each sample) were fragmented to a mean size of 400–500 bp by using a Covaris S220 System (Covaris, Woburn, Massachusetts, USA). Then, an Ion Xpress Plus Fragment Library Kit (Life Technologies) was employed to prepare barcoded shotgun libraries. To conduct emulsion PCR, an Ion PGM Template OT2 400 Kit (Life Technologies) was utilized. Sequencing was performed by the Ion Torrent PGM (Life Technologies) analyser using Ion 318 chips and Ion PGM sequencing 400 Kit v2 (Life Technologies) in accordance with the manufacturer’s protocol.

Babenko V.V., Podgorny O.V., Manuvera V.A., Kasianov A.S., Manolov A.I., Grafskaia E.N., Shirokov D.A., Kurdyumov A.S., Vinogradov D.V., Nikitina A.S., Kovalchuk S.I., Anikanov N.A., Butenko I.O., Pobeguts O.V., Matyushkina D.S., Rakitina D.V., Kostryukova E.S., Zgoda V.G., Baskova I.P., Trukhan V.M., Gelfand M.S., Govorun V.M., Schiöth H.B, & Lazarev V.N. (2020). Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches. BMC Genomics, 21, 331.

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Metagenomic Approach to Identify Metal Tolerance Genes

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Total RNA was isolated from soil samples by using PowerSoil® Total RNA Isolation Kit (Mo Bio laboratories, Carlsbad, CA). Integrity of the RNA was verified through gel electrophoresis and with Bioanalyzer 2100 (Agilent Technologies, USA). cDNAs were synthesized from total soil RNA with Mint-2 cDNA synthesis kit (Evrogen, Moscow, Russia). Size fractionation of cDNAs was performed as described in Yadav et al. (2014) (link). Size fractions of cDNAs, A (<0.5 kb), B (0.5-1 kb) and C (>1 kb) were ligated downstream of PGK1 promoter of S. cerevisiae shuttle plasmid vector pFL61 modified with SfiA and SfiB restriction sites (Minet et al. 1992 ). These recombinant plasmids were introduced into DH10 β electrocompetant E. coli cells (One Shot® TOP10 Electrocomp™ E. coli, Invitrogen). More than 10 6 colonies growing on ampicillin-containing Luria agar plates (100 μg ampicillin per ml of medium) were pooled to represent each of the cDNA size libraries. The protocol used for isolation of metal tolerance genes through metatranscriptomic approach has been summarized in Fig. 1.
Fig. 1 Overview of the protocol used for isolation of genes involved in metal tolerance by metatranscriptomic approach

Mukherjee A., Thakur B., Pandey A.K., Marmeisse R., Fraissinet-Tachet L, & Reddy M.S. (2021). Multi-metal tolerance of DHHC palmitoyl transferase-like protein isolated from metal contaminated soil. Ecotoxicology (London, England), 30(1).

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Transcriptome Sequencing and Analysis

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Isolated total RNA was sent to the North Carolina State University Genomic Sciences Laboratory for library preparation and sequencing. RNA quality and concentration were first checked on the Agilent Bioanalyzer 2100 (Agilent Technologies). About 2 μg of total RNA was used for cDNA library preparation using a combination of three kits-Mint-2 cDNA Synthesis Kit (SK005, Evrogen), Trimmer Direct cDNA Normalization Kit (NK002, Evrogen) and GS FLX Titanium Rapid Library Preparation Kit (05608228001, Roche) according to the manufacturer’s protocol. The library was run on the Roche GS FLX (Roche Applied Science) and sequenced. The data was generated using GS De Novo Assembler software (Roche Applied Science). The data were summarized in Supplementary Table S1a.

Hung C.Y., Qiu J., Sun Y.H., Chen J., Kittur F.S., Henny R.J., Jin G., Fan L, & Xie J. (2016). Gibberellin deficiency is responsible for shy-flowering nature of Epipremnum aureum. Scientific Reports, 6, 28598.

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Quantitative Real-Time PCR Analysis of TAMRA+ and TAMRA- Subpopulations

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HH47 and Krebs-2 ascites tumor cells were sorted into TAMRA+ and TAMRA− subpopulations, and RNA was isolated. CDNA was synthesized for each group using a Mint-2 cDNA synthesis kit (Evrogen, Moscow, Russia). PCR primers for coding regions of each gene were designed using Vector NTI v.9 (Life Technologies, Wilmington, DE, USA) software (Table S1). The primers were tested for specificity using the total cDNA as a template. Real-time qPCR was run on a CFX Connect real-time PCR detection system (BioRad Inc., Hercules, CA, USA) using BioMaster HS-qPCR Hi-Rox SYBR Blue (2×) reagent (Biolabmix, Novosibirsk, Russia). Murine Actb and human Rplp0 genes were used as references.
The cycling parameters were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, with a final melting step with slow heating from 6 °C to 95 °C. All reactions were run in triplicate within a single run, and negative control reactions without reverse transcription reaction and template were also performed. A total of six independent sorting procedures were performed, followed by RNA extraction and cDNA synthesis for real-time PCR for Krebs-2 cells, with four procedures performed for HH47.

Petrova D.D., Dolgova E.V., Proskurina A.S., Ritter G.S., Ruzanova V.S., Efremov Y.R., Potter E.A., Kirikovich S.S., Levites E.V., Taranov O.S., Ostanin A.A., Chernykh E.R., Kolchanov N.A, & Bogachev S.S. (2022). The New General Biological Property of Stem-like Tumor Cells (Part II: Surface Molecules, Which Belongs to Distinctive Groups with Particular Functions, Form a Unique Pattern Characteristic of a Certain Type of Tumor Stem-like Cells). International Journal of Molecular Sciences, 23(24), 15800.

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