The structure of the HvCMF3 gene was determined by analysis of its cDNA. Total RNA was extracted from leaf material of a 3-day-old barley seedling (cv. Barke) using the Trizol reagent (Thermo Scientific, Wilmington, DE, United States) following the manufacturer’s instructions. Concentration of the RNA is measured by help of a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, United States) and further diluted to 1 μg/μL for downstream application. The prepared RNA was first treated with RNase-free DNase I (Fermentas, St. Leon-Rot, Germany) to remove potential DNA contamination; then used for cDNA synthesis applying the SuperScriptTM III First-Strand Synthesis System Kit (Thermo Scientific, Wilmington, DE, United States) following the manufacturer’s instructions. Next, RT-PCR was performed using primers that cover the HvCMF3 coding regions (Supplementary Table 1 ) as previously described (Li et al., 2019 (link)). RT-PCR products were purified using the NucleoFast® 96 PCR Kit (Macherey-Nagel, Düren, Germany) and Sanger sequenced on an ABI 3730 XL platform (Life Technologies GmbH, Darmstadt, Germany). The HvCMF3 exon-intron-structure was revealed by alignment of the coding sequence to the corresponding genomic region.
Superscripttm 3 first strand synthesis system kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SuperScript™ III First-Strand Synthesis System Kit is a reverse transcription kit used for the synthesis of first-strand cDNA from RNA templates. It contains the necessary components to perform reverse transcription reactions, including the SuperScript III reverse transcriptase enzyme, reaction buffer, and other reagents.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Nucleofast 96 pcr kit, by Macherey-Nagel (1 mentions)
Abi3730xl platform, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (1 mentions)
Power sybrtm green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Trizoltm reagent, by Thermo Fisher Scientific (1 mentions)
Cfx96tm real time pcr, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Easy spin total rna extraction kit, by iNtRON Biotechnology (1 mentions)
Gel documentation, by Syngene (1 mentions)
6 protocols using superscripttm 3 first strand synthesis system kit
1
Determining the Structure of the HvCMF3 Gene
2
Quantitative Real-Time PCR Gene Expression Analysis
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Total RNA was extracted from the cells and tissues using the TRIzolTM Reagent (ThermoFisher Scientific, #15596026) according to the manufacturer's instructions. A total of 0.5 μg RNA from each sample was subjected to reverse transcription to obtain cDNA using a SuperScriptTM III First-Strand Synthesis System Kit (ThermoFisher Scientific, #18080051). The resulting cDNA was diluted 100-fold and applied to a qRT-PCR assay using the Power SYBRTM Green PCR Master Mix (ThermoFisher Scientific, #4367659) with the primers listed in Supplementary Table 2 . The expression of β-actin was used for normalization using the 2-ΔΔCt method.
3
Yeast gene expression analysis by qPCR
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RNA was extracted from ~4.5 × 107 yeast cells by glass bead beating using the RNeasy mini kit (QIAGEN). Five hundred nanograms of total RNA was used as starting material for cDNA synthesis using SuperScriptTM III First-Strand Synthesis System Kit (Thermo Fisher Scientific; Cat. No. 18080051). Quantitative real-time PCR was performed on CFX96TM Real-Time PCR (Bio-Rad) in a 96-well plate. Twenty microliters of PCR reactions were prepared with 2X mastermix and 20X Taqman assay (PUT6 assay ID: Sc04120572_s1, PUT7 assay ID: Sc04148698_s1, PUT1 assay ID: Sc04147047_s1, and ACT1 assay ID: Sc04120488_s1) from Applied Biosystems. The mRNA levels were normalized to ACT1 expression levels.
4
Genomic DNA and RNA Extraction and qRT-PCR Analysis
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The genomic DNA was extracted through a quick and safe method for screening of transformants (Chi et al., 2009 ). Genomic DNA used in Southern blotting was extracted through a standard method (Park et al., 2013 (link)). Total RNA was isolated from the frozen fungal tissues using Easy-Spin Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea). The cDNA was synthesis using SuperScriptTM III First-Strand Synthesis System Kit (Invitrogen Life Technologies, Carlsbad, CA, USA) (Shin et al., 2022 (link)). Quantitative reverse transcription PCR was performed with Real-Time PCR 2× Master Mix (Elpis Bio, Daejeon, Korea) using a StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) (Fu et al., 2021 (link)). The target gene expression was expressed as 2−ΔΔCt, where ΔΔCt = (Ct, target gene − Ct, β-tubulin) control − (Ct, target gene − Ct, β-tubulin) test condition (Livak and Schmittgen, 2001 (link)). This experiment was performed in three-independent experiments with two replicates per experiment.
5
Total RNA Isolation and cDNA Synthesis
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Total RNA was extracted from cryopreserved OTs using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) as described previously (Kleszczyński et al., 2016 (link)). The yield of total RNA for each sample was quantified with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). First-strand cDNA of the total RNA was synthesized by reverse transcription using the SuperScriptTM III First-Strand Synthesis System Kit (Invitrogen, Carlsbad, CA, United States).
6
RNA Isolation and RT-PCR Analysis
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The isolation of total RNA was performed from harvested cells using Trizol® Reagent (Invitrogen, USA). The first stand cDNA synthesis was used in one microgram of total isolated RNA. The reaction was performed by SuperScriptTM III First-Strand Synthesis System kit (Invitrogen, USA). RT-PCR amplifications using cDNAs of the respective genes were performed using corresponding primers listed in Table 3 . The PCR reaction was consisted of initial denaturation at 98 °C for 30 sec, followed by by 29 cycles for PlsC and other genes studied, and 14 and 26 cycles for 16 s and PlsX, respectively, of three steps including denaturation at 94 °C for 10 sec, annealing step of each specific pair of primers at 55 °C for 30 sec and extension at 72 °C for 25 sec, followed by final extension at 72 °C for 5 min. The PCR products were analyzed by 0.8% (w/v) agarose gel electrophoresis and quantification was done using Syngene® Gel Documentation (Syngene, Frederick, MD).
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