For analysis of candidate gene expression in leaves, ten each of the alkalinity-tolerant and alkalinity-sensitive rice accessions were selected according to the SAT of 295 japonica rice accessions. The procedure and management of the experiment were the same as those in the above-mentioned experiment. After 48 h of alkalinity stress implemented with 0.15% of Na2CO3, five shoots from each variety were sampled under alkaline and normal conditions. Total RNA was extracted from rice shoots using a TranZol Up RNA kit (TransGen Biotech). All samples were treated with DNase I (TransGen Biotech). Complementary DNA was synthesized from total RNA using HiFiScript cDNA Synthesis Kit (CoWin Biosciences, Beijing, China). qRT-PCR was performed using 2 × Fast qPCR Master Mixture (DINING, Beijing, China) on Analytik Jena qTOWER system (German). Additional file 8 : Table S6 summarizes the gene accessions and primers used for qRT-PCR in this study. The mRNA level of these genes was determined with the housekeeping gene Actin1 (Li et al. 2018 (link)) as an internal control. Relative gene expression levels were determined using the 2 −ΔΔCt method (Livak and Schmittgen 2001 (link)). The data shown in figures and tables are mean values of three replicates.
Dnase 1
Manufactured by Transgene
Sourced in China
DNase I is a laboratory enzyme used to degrade DNA. It functions by cleaving the phosphodiester bonds in DNA molecules, breaking them down into smaller fragments. This enzyme is commonly used in various molecular biology and cell biology procedures, where the removal of DNA is necessary.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Easyscript first strand cdna synthesis supermix, by Transgene (3 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Mastercycler ep realplex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Bestar sybrgreen qpcr mastermix, by DBI Bioscience (2 mentions)
Transscript first strand cdna synthesis supermix, by Transgene (2 mentions)
Trizol, by Tiangen Biotech (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Hifiscript cdna synthesis kit, by CoWin Biotech (1 mentions)
Tranzol up rna kit, by Transgene (1 mentions)
Qtower system, by Analytik Jena (1 mentions)
Lysozyme, by Avantor (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Solarbio (1 mentions)
Dna polymerase, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Rutin, by Solarbio (1 mentions)
Sybr green real time pcr master mix, by Vazyme (1 mentions)
28 protocols using dnase 1
1
Quantifying Alkalinity-Responsive Gene Expression
2
Mycotoxin Detection and Enzyme Purification
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AFB1, ZEN, DON, and the commercial Nematoloma frowardii manganese peroxidase (NfMnP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). FB1 and OTA were purchased from Pribolab (Beijing, China). Hemin was purchased from TCI (Tokyo, Japan). DNA polymerase, T4 ligase, and chromatographic grade reagents (acetonitrile, methanol, methanoic acid, acetic acid, and trifluoroacetic acid) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). DNase I and TransScript One-Step gDNA Removal and cDNA Synthesis Supermix with oligo(dT) were purchased from TransGen (Beijing, China). Isopropyl-β-D-thiogalactoside (IPTG), SOD, DTT, and Rutin were purchased from Solarbio (Beijing, China). TRIZOL was from Invitrogen (Carlsbad, CA, USA). Lysozyme was purchased from Amresco (Solon, OH, USA). Ni-NTA agarose was purchased from QIAGEN (Duesseldorf, Germany). All other chemicals were of analytical grade or chromatographically pure, and were commercially available.
3
RNA Extraction and qRT-PCR Analysis of Arabidopsis
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The total RNA was extracted from the frozen Arabidopsis leaf samples with RNAiso Plus (Takara 9101). The resulting RNA samples were treated with DNase I (TransGen Biotech GD201-01) followed by reverse transcription using HiScript II Reverse Transcriptase (Vazyme R223-01) according to the manufacturer’s instructions. qRT-PCR was performed in triplicate using SYBR Green Real-Time PCR Master Mix (Vazyme Q711-02) on the Bio-Rad CFX96 system. The 2− ∆∆ Ct method was used to calculate the relative gene expression level across the samples. Finally, the results were presented as histograms by GraphPad Prism 8 software (GraphPad, San Diego, CA, USA). Primers used are listed in Table S3 .
4
Transcriptome Analysis of H. axyridis
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Five days after oviposition, the total RNA from H. axyridis adult females was extracted using the Tranzol reagent according to the manufacturer’s instructions. The control and treatment group samples were collected from the first abdominal ganglia to the tail end. Genomic DNA was digested with DNase I (Transgene, Beijing, China), and the RNA integrity was determined by agarose gel electrophoresis, which revealed clear bands for 18S and 28S. For ligation to sequence adapters, the cDNA was purified and repaired, and A bases were added to the 3′-ends.
5
Radish Hypocotyl RNA Extraction and qRT-PCR
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Freshly harvested radish hypocotyls (100 mg) were adequately ground to powder with liquid nitrogen for RNA extraction. Total RNA was extracted using the Trizol reagent (Invitrogen, Gaithersburg, MD) following manufacturer’s instruction, and finally dissolved in 50 μl RNase-free water. The extracted RNA was disposed with DNase I (RNase-free, Transgen®) at 25 °C for 30 min, followed by performance of reverse transcription according to the manufacturer’s instruction (TransScript® First-Strand cDNA Synthesis SuperMix, Transgen®). The qRT-PCR reactions were manipulated by utilizing a Mastercycler®ep realplex real-time PCR system (ABI7500, MD, USA) with Bestar® SybrGreen qPCR mastermix (DBI, Bioscience Inc.,Germany) in a 20 μL reaction volume. The primers as shown in Table S2 .
6
Quantitative PCR Analysis of NMD Genes
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Approximate 10–20 wandering L3 larvae were collected and RNA was extracted by TransZol Up (TransGen Biotech, cat#ET111-01). DNase I (TransGen Biotech, cat#GD201-01) was used to digest genomic DNA. cDNA was synthesized with iScript Reverse Transcription kit (Bio-Rad, cat#1708890) and iTaq Universal SYBR Green Supermix (Bio-Rad, cat#1725120) was used for quantitative PCR. Analysis was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad). rp49 was used as a reference gene69 (link),70 (link). All PCR reactions were performed in biological triplicate. Primers used were:
rp49-For: 5′-GGCCCAAGATCGTGAAGAAG-3′;
rp49-Rev: 5′-ATTTGTGCGACAGCTTAGCATATC-3′;
upf1-For: 5′-ACTTCCGGTTCGCACATCAT-3′;
upf1-Rev: 5′-CTTCCACTGTTCCTGGTCCC-3′;
upf3-For: 5′- ATGCTCCCTTCCAGTGCTTC-3′;
upf3-Rev: 5′- CCGCTTGATGAACTCCTGGT-3′;
smg5-For: 5′- GCTTTTTGACTGGCTGCGAA-3′;
smg5-Rev: 5′- ACCAGAGAATCACGCACGTT-3′;
nluc-For: 5′-GATCATCCCCTACGAGGGCT-3′;
nluc-Rev: 5′-GTCGATCATGTTGGGGGTCA-3′;
rab6-3′UTR-For: 5′-ATCCAACCATCCTCTCCCCC-3′;
rab6-3′UTR-Rev: 5′-GCAGATCCGGCCAGTACATA-3′;
rps20-3′UTR-For: 5′- ATCATCGACTTGCACTCGCC-3′;
rps20-3′UTR-Rev: 5′- GCACGCCAAACTTTTCGAGG-3′.
rp49-For: 5′-GGCCCAAGATCGTGAAGAAG-3′;
rp49-Rev: 5′-ATTTGTGCGACAGCTTAGCATATC-3′;
upf1-For: 5′-ACTTCCGGTTCGCACATCAT-3′;
upf1-Rev: 5′-CTTCCACTGTTCCTGGTCCC-3′;
upf3-For: 5′- ATGCTCCCTTCCAGTGCTTC-3′;
upf3-Rev: 5′- CCGCTTGATGAACTCCTGGT-3′;
smg5-For: 5′- GCTTTTTGACTGGCTGCGAA-3′;
smg5-Rev: 5′- ACCAGAGAATCACGCACGTT-3′;
nluc-For: 5′-GATCATCCCCTACGAGGGCT-3′;
nluc-Rev: 5′-GTCGATCATGTTGGGGGTCA-3′;
rab6-3′UTR-For: 5′-ATCCAACCATCCTCTCCCCC-3′;
rab6-3′UTR-Rev: 5′-GCAGATCCGGCCAGTACATA-3′;
rps20-3′UTR-For: 5′- ATCATCGACTTGCACTCGCC-3′;
rps20-3′UTR-Rev: 5′- GCACGCCAAACTTTTCGAGG-3′.
7
Quantitative Real-Time PCR of Arabidopsis Seedlings
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Total RNA was extracted from one-day germinated seeds or 7-day-old Arabidopsis seedlings using the EasyPure Plants RNA Kit (TransGen, Beijing, China). After DNA depletion by DNase I (TransGen, Beijing, China), 1 μg total RNA was used to synthesize cDNA using ReverTra Ace qPCR RT Master Kit (Toyobo Co., Ltd., Osaka, Japan). Quantitative real-time PCR analysis was performed using SYBR Premix Ex Taq (Takara, Tokyo, Japan) in an ABI 7500 fast real-time instrument (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The relative expression levels were normalized to internal control ACTIN2. The qRT-PCR assays were performed with three biological replicates, and three technical replicates were performed in each biological replicates. Information regarding the primers used in this assay is available in S7 Table .
8
RNA Extraction and cDNA Synthesis from Insect Tissues
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Total RNA was extracted from the antennae, heads, abdomens, thoraxes, and legs using Trizol Reagent (Invitrogen, Carlsbad, CA, United States). The RNA was quantified and assayed for purity using gel electrophoresis and NanoDrop ND-2000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, United States), and treated with DNaseI (TransGenBiotech, China) to remove trace amounts of genomic DNA. The first single-strand cDNA synthesis used RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States) and served as a template in PCR or RT-PCR reactions.
9
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from the above tissues using Trizol Reagent (Invitrogen, Carlsbad, CA, United States). The NanoDrop ND-2000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, United States) were used to verify the purity and concentration of the RNA, and gel electrophoresis was used to verify the integrity of RNA. Total RNA was treated with DNaseI (TransGenBiotech, China) to remove traces of genomic DNA. Total RNA (1 μg) of the different tissues were used for first single-strand cDNA synthesis used RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, United States). The resultant cDNA was used as the templates for gene clone and Real-time quantitative PCR (qPCR). The qPCR experiments were repeated three times using three independently isolated RNA samples.
10
Quantitative RT-PCR for Gene Expression
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Total RNA was extracted with Trizol (Invitrogen). Total RNA was treated with DNase I (Transgen, Beijing, China). The first-strand cDNA was generated by reverse transcription with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China) by Oligo(dT) RTprimer (mRNAs) according to the manufacturer’s instructions. For regular PCR reactions, PCR products were fractioned in 1% agarose gels. For qRT-PCR, the amplification systems were processed in a 20 μL solution with 10 μL TransStart Green qPCR SuperMix (Transgen, Beijing, China), 4 ng template, and 0.8 μL (10 μmol) primers (Table S8 ). The reactions were run at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing at 60 °C for 1 min in the 7500 Real-Time PCR System (Applied Biosystems) [16 (link)]. Brassica Actin2 serves as an internal control. All primers used are listed in Table S8 (Table S8 ). Primers used for genotyping were designed on http://signal.salk.edu/tdnaprimers.2.html (accessed on 14 December 2022). AtRH6-qRT primers were synthesized according to [25 (link)]. AtActin2 primers were synthesized according to [19 (link)]. BnActin2 primers were synthesized according to [92 (link)]. Other primers were designed with Primer Premier6.
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