Total RNA was extracted from leaves of 9311 plants and spl 30 mutants with TRIzol reagent (Invitrogen, USA). The first-strand synthesis of cDNA was done with a TransScriptTM one-step gDNA removal and cDNA synthesis SuperMix (TransGen, China) according to the manufacturer’s instructions. The first-strand cDNA was used as a template for amplification by PCR (30 cycles for spl5, spl11 and spl28 and 25 cycles for the actin control). The reaction mixture was cycled through the following temperature profiles: 94 °C for 210 s for one cycle, followed by 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 60 s for 30 cycles, and a final incubation at 72 °C for 5min. The primers for RT-PCR were as follows: spl5-Forward (5′-TTGCAG CAAACTTCATCAGGAC-3′) and spl5-Reverse (5′-GAGGGACTCCAAGGAAAGTGTTAT-3′) for spl5, spl11-Forward (5′-GATGCTTGCCTTATTGTCCTCA--3′) and spl11-Reverse (5′-ACGGATTGATATGCCTGA CGAT-3′) for spl11, spl28-Forward (5′-GTGAAAGCA AGAAGTCAGTTTAAGG-3′) and spl28-Reverse (CTAACAAGATGAACAACGAGACAGA-3′) for spl28, actin-Forward (5′-TGTCATGGTTGGAATGGGCCA-3′) and actin-Reverse (5′-AGGCAGTCAGTCAGATCA CGA-3′) for actin. Each analysis was repeated independently three times using different biological samples each time.
Transscript one step gdna removal and cdna synthesis supermix
Manufactured by Transgene
Sourced in China, United States
TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix is a laboratory product that performs two functions in a single reaction: removal of genomic DNA (gDNA) and synthesis of complementary DNA (cDNA) from RNA samples. This all-in-one solution streamlines the RNA-to-cDNA conversion process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (63 mentions)
Transstart top green qpcr supermix, by Transgene (34 mentions)
Perfectstart green qpcr supermix, by Transgene (17 mentions)
Trizol, by Thermo Fisher Scientific (16 mentions)
Transstart tip green qpcr supermix, by Transgene (15 mentions)
Sybr green realtime pcr master mix, by Toyobo (12 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (11 mentions)
Transzol up plus rna kit, by Transgene (11 mentions)
Eastep super total rna extraction kit, by Promega (11 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (10 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (10 mentions)
Easypure plant rna kit, by Transgene (9 mentions)
Lightcycler 96, by Roche (8 mentions)
Rnaiso plus, by Takara Bio (8 mentions)
Easypure rna kit, by Transgene (8 mentions)
Rnaprep pure micro kit, by Aidlab (7 mentions)
Chamq sybr qpcr master mix, by Vazyme (7 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (7 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Sybr premix ex taq 2, by Takara Bio (6 mentions)
407 protocols using transscript one step gdna removal and cdna synthesis supermix
1
RNA Extraction and RT-PCR Analysis of SPL Genes
2
Quantitative Real-Time PCR Analysis of Transgenic Plants
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For real time qRT-PCR analysis, samples were harvested from the shoot apex of 45-day-old seedlings of T1 transgenic tobacco plants or 3-week-old seedlings of transgenic Arabidopsis plants. Three biological replications were performed randomly for each transgenic line. Total RNA was isolated using Trizol reagent (Takara) according to the manufacturer’s instructions. The first strand of cDNA was synthesized using 2 μg of total RNA as a template with the TransScriptTM one-step gDNA Removal and cDNA Synthesis Supermix (Transgen, Beijing, China). The qRT-PCR was performed on 7500 Fast Real-Time PCR System (Applied Biosystems) with SYBR Premix EX TagTM (Takara). The tobacco NtEF1α and Arabidopsis AtEF1α transcript were used as an internal standard to calculate the relative expression by the comparative CT (△△CT) method, respectively. The primers for RT-PCR and qRT-PCR are detailed in Supplementary Tables S5 , S6 .
3
Transcriptome Analysis of Plant Stress Response
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Total RNA was extracted from the frozen plant material using the TRIzol reagent (Invitrogen, USA) following to the manufacturer’s instructions. The resulting RNA was treated with RNase-free DNaseI (Promega, USA) to remove genomic DNA contamination, and the cDNA First strand was synthesized with 3 μg total RNA by TransScript One-step gDNA Removal and cDNA synthesis SuperMix (TransGen, China) following the manufacturer’s protocol. The subsequent RT-qPCRs and data analysis were performed using a Bio-Rad Real-Time PCR detection system (Bio-Rad) based on the SYBR Green I master mix, as reported previously (Bustin et al. 2009 (link); Seo et al. 2009 (link)). According to a previous study, GmELF1b was most stably expressed under salt stress, so it was used as a reference gene (Le et al. 2012 ). All reactions were carried out in triplicate, using samples harvested from independent plants. The relevant primer sequences are given in Additional file 1 : Table S1.
4
Quantifying Bacillus subtilis Promoter Activity
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To determine the promoter’s activity, total RNA was isolated from 18 h cultures of the transformed B. subtilis strains EGFP-WT, EGFP-T2G, EGFP-T2C and EGFP-T3C, harboring the recombinant plasmids Pylb-G-P43-R-pUBC19, pGT2G, pGT2C, and pGT3C, using a RNAprep PureCell/Bacteria Kit (TIANGEN Biotech, Beijing, China). The first strand of cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) with the gene-specific primers egfp-rtR and BS16S-rtR (Supplementary Table 2). The qRT-PCR was performed using the SYBR Green Real-time PCR Master Mix Plus (Toyobo, Osaka, Japan) with the primers egfp-rtF and egfp-rtR (Supplementary Table 2) for the egfp gene and primers BS16S-rtF and BS16S-rtR (Supplementary Table 2) for the 16SrDNA gene.
5
Transcriptional Analysis of TaOSCA1.4 in Wheat
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The transcript levels of TaOSCA1.4 in Fielder organs were analyzed via qRT−PCR. The primers used were as follows:
The first-strand cDNAs were synthesized from 2 µg of RNA per sample using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). The qRT−PCR aplifications were performed as described by Zhao et al. (2020) (link). Amplification of TaActin was used as an internal control for data normalization. The experiments were independently replicated three times under identical conditions. The complete alignment of multiple coding sequences and translations of nucleotides into amino acid sequences were performed using the DNAMAN program (version 5.2.2; Lynnon Biosoft, Canada). The prediction of the transmembrane structure was performed using DeepTMHMM (https://dtu.biolib.com/DeepTMHMM ). Collinearity analysis was performed using the Triticeae-Gene Tribe (TGT, http://wheat.cau.edu.cn/TGT/ ).
The first-strand cDNAs were synthesized from 2 µg of RNA per sample using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). The qRT−PCR aplifications were performed as described by Zhao et al. (2020) (link). Amplification of TaActin was used as an internal control for data normalization. The experiments were independently replicated three times under identical conditions. The complete alignment of multiple coding sequences and translations of nucleotides into amino acid sequences were performed using the DNAMAN program (version 5.2.2; Lynnon Biosoft, Canada). The prediction of the transmembrane structure was performed using DeepTMHMM (
6
Real-time qRT-PCR analysis of StUSP genes
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BIOER’s LineGene 9620 real-time fluorescence quantitative PCR system was used to select 10 genes for qRT-PCR analysis, and β-action was used as the reference gene (Table S11 ). These genes included StUSP2, StUSP6, StUSP13, StUSP14, StUSP15, StUSP21, StUSP24, StUSP9, StUSP33, and StUSP41; the primer sequences were designed using prime-blast [89 (link)]. Total RNA was extracted from each treated sample using the Total RNA Rapid Extraction Kit (ER501-01, TransGen Biotech, Beijing, China) and reverse-transcribed to cDNA using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311, TransGen Biotech, Beijing, China). qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China) kit. Three biological replicates were calculated using the 2−∆∆Ct method [90 (link)].
7
Transcriptional Analysis of Fungal Mutants
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Total RNA extraction and qRT-PCR analysis were performed as described by Guan et al. (55 (link)). Total RNA of the mycelia of the HN08 strain and mutants were extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). cDNA synthesis was performed with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). The expression levels of the target genes were quantified by qRT-PCR performed with an ABI7500 sequence detection system (Applied Biosystems, Waltham, MA, USA). Reactions were performed in a total volume of 10 µL using the SYBR Premix Dimer Eraser Kit (Takara, Beijing, China). All of the reactions were repeated in at least three independent pools in three sets of biological replicates. The primer sequences were listed in Table S1 .
8
Uterine Gene Expression Analysis
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The expression of keratin, vimentin, integrin β3, IL-2, TNFα, IFN-γ, IL-4, IL-10, VEGF, MMP9, and Ki-67 was assessed. Total RNA from rat uteri was isolated using TRI Reagent (Sigma) according to the manufacturer's protocol. Complementary DNAs (cDNAs) were synthesized using Trans Script One-Step gDNA Removal and cDNA Synthesis Super Mix (TransGen Biotech, Beijing, China). Real-time quantitative PCR was performed using primers and the Universal KAPA SYBR FAST qPCR Kit (Roche, Switzerland) in a 7900 Real-Time PCR System (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. All experiments were repeated at least three times. Primer sequences used in real-time quantitative PCR were showed in Table S2 .
9
RNA Extraction and qRT-PCR Analysis
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BmN cells were harvested at the corresponding time points and the total RNA was extracted using the TRIzol™ reagent ((TaKaRa, Dalian, China). Thereafter, the first-strand cDNAs was synthesized using 5 μg total RNA with the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). The primers are listed in Supplementary Table S2 . qRT-PCR was performed using Hieff® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). 95 °C for 5 min, then 40 cycles at 95 °C for 5 s, and 60 °C for 30 s were included in the reaction programs. Each sample was triplicated. The 2−ΔΔCt method was applied to calculate the relative expression levels of selected genes.
10
TRIzol RNA Extraction and cDNA Synthesis
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Total RNA from the samples was extracted using the TRIzol (Trizol reagent 10296028, United States) method and the reverse transcription kit (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, AT311-03, TransGen Biotech, China) was used to synthesize first-strand cDNA and remove genomic DNA. Total RNA was stored in a −80 ± 1°C refrigerator prior to subsequent experimentation.
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