Quantitative real-time polymerase chain reaction was performed to quantify the expression levels of METTL14 and FTO using Green Premix Ex Taq II (TaKaRa Bio) and CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The cycle parameters were 95°C for 5 minutes, 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds, and 65°C for 5 seconds. METTL14 quantitative real time polymerase chain reaction primers used were as follows: forward, 5′-AGTGCCGACAGCATTGGTG-3′ and reverse, 5′-GGAG- CAGAGGTATCATAGGAAGC-3′. FTO quantitative real-time polymerase chain reaction primers used were: forward, 5′-TTGGACGGTACAGATATGGAACATTTT-3′ and reverse, 5′-TCTTTTAGTTTCT-TTGCCTTTGGGGAT-3′. mRNA levels were normalized to reference gene sequences of β-actin forward, 5′-CTGGAACGGTGAAGGTGACA-3′ and reverse, 5′-AAGGGACTTCCTGTAACAATGCA-3′. The absolute expression levels of METTL14 and FTO were calculated using the 2−ΔΔCt method.
Green premix ex taq 2
Manufactured by Takara Bio
Sourced in China
Green Premix Ex Taq II is a ready-to-use PCR master mix that contains all the necessary components for DNA amplification, including DNA polymerase, dNTPs, and reaction buffer. It is designed to simplify the PCR setup process and improve efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Revertaid cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rnase free dnase 1, by Takara Bio (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Cfx connected real time system, by Bio-Rad (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Origin 9, by OriginLab (1 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
7 protocols using green premix ex taq 2
1
Quantitative Analysis of METTL14 and FTO
2
Quantitative Gene Expression Analysis
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The hub genes were verified by Quantitative Reverse Transcription-PCR (qRT-PCR). Total RNA was reverse-transcribed to cDNA using First Strand cDNA Synthesis Kit (Life Technologies, Carlsbad, CA, United States) according to the manufacturer’s instructions. A CFX connected Real-time System (Bio-Rad, Hercules, CA, United States) and Green Premix ex Taq II (Takara, Dalian, China) was used for quantitative PCR. All primers used in this study are listed in Supplementary Table S1 . All samples were normalized to GAPDH. And the relative expression levels of each gene were calculated using 2−ΔΔCt methods.
3
Validation of Terpenoid Pathway Genes in Rosa chinensis
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Stage 5 petals and different tissues from OB and OBL were collected and processed for total RNA extraction and first-strand cDNA synthesis as previously described [51 (link)]. The key DEGs involved in the terpenoid metabolic pathway and RcWRKY70 were subjected to validation by RT–qPCR using RcACTIN as the reference transcript. Primer sequences are provided in Table S11 . qPCR was performed with a Green® Premix Ex Taq™ II (RR420Q, Takara, Tokyo, Japan) and the CFX Connect Real-Time PCR Detection System (Bio–Rad, Hercules, CA, USA). Each sample was tested as three technical replicates. Relative gene expression levels were obtained by the comparative 2−ΔΔCt method [52 ]. All results were plotted in Origin9 (OriginLab, Northampton, MA, USA).
4
Arabidopsis Transgenic RNA Extraction and Analysis
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Total RNA was extracted from the whole transgenic Arabidopsis plant using RNAprep Pure Plant Kit. RNA reverse transcription was performed using HiScript® III RT SuperMix for semi-qRT-PCR and qRT-PCR using primer references (Table 1 ). Semi-qRT-PCR was performed using ABI VeritiPro PCR (Thermo Fisher). Semi-qRT-PCR analysis was performed in a total volume of 25 µL containing 12.5 μL Green™ Premix Ex Taq™ II (TaKaRa), 0.5 μL of each primer, 1 μL cDNA, and 10.5 μL nuclease-free water. The GlPS1 and GlActin gene-specific primers used for semi-qRT-PCR are shown in Table 1 . Statistical data were evaluated by Student’s t test.
5
Quantitative Analysis of Colorectal Cancer Transcriptome
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Thirty pairs of CRC tumor and adjacent tissues were collected from clinical patients at The First Affiliated Hospital of Wenzhou Medical University and preserved in -80 C refrigerator. All patients gave the written informed consent. All assay regimens gained the approval of the Ethics Committees in Clinical Research of the First Affiliated Hospital of Wenzhou Medical University and the corresponding ethical approval code was KY2021-R005. Total RNA was isolated from tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA). Then PrimeScript™ RT Master Mix kit (TaKaRa, Japan) kit was used to synthesize cDNA according to the instruction manual. q-PCR for cDNA amplification was performed with Green™ Premix Ex Taq™ II (TaKaRa) kit. GAPDH was used as endogenous control and primers were shown in Supplementary Table S1 . The expression of signature DE-FLs were normalized using the relative quantification method of 2-ΔΔCt.
6
Quantifying ABA and Stress Response in Tobacco
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To assess the transcript levels of ABA and stress-responsive genes in tobacco seedlings, quantitative real-time PCR (qPCR) was employed. For this, two weeks old seedlings of wild-type, GhNAC4, GhNAC4-N, GhNAC4-C genotypes were treated with 200 mM NaCl and 15% PEG 8000 for 24 h.
Total RNA was extracted using Trizol reagent (Invitrogen, USA) and DNA contamination was removed by treating it with RNase-free DNase I (Takara Bio, China). Approximately 1 μg of total RNA was used for the first-strand cDNA synthesis using RevertAid cDNA synthesis kit (Thermo Fischer Scientific, USA) according to the manufacturer's instructions. For the qPCR, cDNA was diluted to 100 ng/µl and was mixed with Green Premix Ex Taq II (Takara Bio, China) and amplifications were carried out following the manufacturer's protocol. The constitutively expressing Ubiquitin gene (NtUBI1, GenBank Accession no. U66264.1) from tobacco was used as an internal reference gene. The stress-responsive genes used in the study are
1), NtNCED3 (JX101472.1), NtSOS1 (XM_009789739.1), and NtSUSY (AB055497.1). The primers of stress-associated genes are mentioned in Supplementary Table 2. The fold change was determined using the ΔΔC T method (Livak and Schmittgen 2001) (link). The experiments were performed in triplicates, and two independent biological replicates were used for the analyses.
Total RNA was extracted using Trizol reagent (Invitrogen, USA) and DNA contamination was removed by treating it with RNase-free DNase I (Takara Bio, China). Approximately 1 μg of total RNA was used for the first-strand cDNA synthesis using RevertAid cDNA synthesis kit (Thermo Fischer Scientific, USA) according to the manufacturer's instructions. For the qPCR, cDNA was diluted to 100 ng/µl and was mixed with Green Premix Ex Taq II (Takara Bio, China) and amplifications were carried out following the manufacturer's protocol. The constitutively expressing Ubiquitin gene (NtUBI1, GenBank Accession no. U66264.1) from tobacco was used as an internal reference gene. The stress-responsive genes used in the study are
1), NtNCED3 (JX101472.1), NtSOS1 (XM_009789739.1), and NtSUSY (AB055497.1). The primers of stress-associated genes are mentioned in Supplementary Table 2. The fold change was determined using the ΔΔC T method (Livak and Schmittgen 2001) (link). The experiments were performed in triplicates, and two independent biological replicates were used for the analyses.
7
Quantifying ABA and Stress Response in Tobacco
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To assess the transcript levels of ABA and stress-responsive genes in tobacco seedlings, quantitative real-time PCR (qPCR) was employed. For this, two weeks old seedlings of wild-type, GhNAC4, GhNAC4-N, GhNAC4-C genotypes were treated with 200 mM NaCl and 15% PEG 8000 for 24 h.
Total RNA was extracted using Trizol reagent (Invitrogen, USA) and DNA contamination was removed by treating it with RNase-free DNase I (Takara Bio, China). Approximately 1 μg of total RNA was used for the first-strand cDNA synthesis using RevertAid cDNA synthesis kit (Thermo Fischer Scientific, USA) according to the manufacturer's instructions. For the qPCR, cDNA was diluted to 100 ng/µl and was mixed with Green Premix Ex Taq II (Takara Bio, China) and amplifications were carried out following the manufacturer's protocol. The constitutively expressing Ubiquitin gene (NtUBI1, GenBank Accession no. U66264.1) from tobacco was used as an internal reference gene. The stress-responsive genes used in the study are
1), NtNCED3 (JX101472.1), NtSOS1 (XM_009789739.1), and NtSUSY (AB055497.1). The primers of stress-associated genes are mentioned in Supplementary Table 2. The fold change was determined using the ΔΔC T method (Livak and Schmittgen 2001) (link). The experiments were performed in triplicates, and two independent biological replicates were used for the analyses.
Total RNA was extracted using Trizol reagent (Invitrogen, USA) and DNA contamination was removed by treating it with RNase-free DNase I (Takara Bio, China). Approximately 1 μg of total RNA was used for the first-strand cDNA synthesis using RevertAid cDNA synthesis kit (Thermo Fischer Scientific, USA) according to the manufacturer's instructions. For the qPCR, cDNA was diluted to 100 ng/µl and was mixed with Green Premix Ex Taq II (Takara Bio, China) and amplifications were carried out following the manufacturer's protocol. The constitutively expressing Ubiquitin gene (NtUBI1, GenBank Accession no. U66264.1) from tobacco was used as an internal reference gene. The stress-responsive genes used in the study are
1), NtNCED3 (JX101472.1), NtSOS1 (XM_009789739.1), and NtSUSY (AB055497.1). The primers of stress-associated genes are mentioned in Supplementary Table 2. The fold change was determined using the ΔΔC T method (Livak and Schmittgen 2001) (link). The experiments were performed in triplicates, and two independent biological replicates were used for the analyses.
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