The lung tissue stored in the refrigerator at –80°C was taken out and cut. After being extracted from the lung tissue using Trizol reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA), total RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (Takara Bio Inc., Osaka, Japan). SYBR® Premix Ex Taq™ (Takara Bio Inc., Osaka, Japan) was used for qRT-PCR to obtain quantitative information on the mRNA expression of HIF-1α, Beclin 1, LC3, ATG5, mTOR, and p62 in the lung tissue. HIF-1α is an important marker of hypoxia and a downstream target of mTOR. Under hypoxic conditions, mTOR expression is upregulated and HIF-1α increases p62 expression, thereby decreasing autophagy. Additionally, the transformation of LC3-I to LC3-II is a marker of autophagosome formation and Beclin 1 of autophagy. The information on primers is provided in Table 1 . The 2−ΔΔCt method was used for the calculation of data, which were presented as normalized expressions relative to glyceraldehyde-3-phosphate dehydrogenase mRNA.
Primescript rt reagent kit with gdna eraser perfect real time kit
Manufactured by Takara Bio
Sourced in Japan, China
The PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) is a set of reagents for reverse transcription and real-time PCR. The kit includes a gDNA Eraser component for the removal of genomic DNA contamination prior to reverse transcription.
Automatically generated - may contain errors
Lab products found in correlation
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Tb green premix ex taq tli rnaseh plus, by Takara Bio (1 mentions)
Midetect a track mirna qpcr kit, by RiboBio (1 mentions)
Midetect a track mirna qrt pcr starter kit, by RiboBio (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Tb green premix ex taq 2 tli rnaseh plus kit, by Takara Bio (1 mentions)
Tb green premix ex tag 2, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Quantstudio 7 flex, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr kit, by Takara Bio (1 mentions)
Pmd19 t vector, by Takara Bio (1 mentions)
Rna extraction kit, by CWBIO (1 mentions)
Sybr premix ex taqtm, by Takara Bio (1 mentions)
Sybr premix ex taqtm 2 tli rnaseh plus kit, by Takara Bio (1 mentions)
10 protocols using primescript rt reagent kit with gdna eraser perfect real time kit
1
Hypoxia-induced autophagy regulatory gene expression
2
Verification of ceRNA Network Expression
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The RNA expressions involved in the ceRNA network in each group were verified by qRT-PCR. For mRNA and lncRNA verifications, PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (TaKaRa, RR047A, Japan) was deployed in reverse transcription procedure. Subsequently, qPCR was conducted, and TB Green® Premix Ex Taq ™ (Tli RNaseH Plus) (TaKaRa, RR420A, Japan) was deployed. The miRNA verification process involved the utilization of the miDETECT A TrackTM miRNA qRT-PCR Starter Kit (C10712, RIBOBIO, China) and the miDETECT A TrackTM miRNA qPCR Kit (C10711, RIBOBIO, China). The quantification of mRNA, lncRNA, and miRNA expression levels was conducted utilizing the 2- ΔΔCT approach. ACTB and GAPDH were used as reference genes for mRNA and lncRNA, U6 and 5S were used as reference genes for miRNA.
3
Validating Transcriptome Sequencing Data
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To verify the accuracy of the transcriptome sequencing results, 20 genes related to biological events such as oocyte meiosis and homologous recombination were selected for qRT-PCR analysis. Total RNA was reversely transcribed into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (Takara, Japan). qRT-PCR reactions were performed using the TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) kit (Takara, Japan). The PCR reaction system contained the following ingredients: 1 μL template cDNA, 10 μl TB Green Premix Ex Taq II (Tli RNaseH Plus)(2 ×), 1 μL upper/downstream primers, and 7 μL RNase Free dH2O. qRT-PCR was performed using the Bio-Rad CFX96 Real-Time System with a cycling program of 30 s at 95 °C; 40 cycles, each at 95 °C for 15 s, at 60 °C for 15 s; at 72 °C for 30 s, and finally at a rate of 0.5 °C every 5 s from 65 °C to 95 °C to generate a melting curve, demonstrating the absence of dimeric primers and generation of non-specific amplification. Ef1-α and β-actin were selected as reference genes. The qRT-PCR experiments were repeated three times for each gene. The relative expression levels of the 20 genes in different samples were calculated using the 2−ΔΔCT method. The primer sequences used are listed in Additional file 1 .
4
Quantitative Gene Expression Analysis in Poplar
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Total RNA was extracted from poplars with a Plant RNA Extraction Kit TRIzol® Reagent (Invitrogen, America, 15 596‐026), and 1 μg of total RNA was used to generate complementary DNA (cDNA) using a PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (TaKaRa, Japan). Gene expression levels were analysed by qPCR with TB Green Premix Ex Tag II (TaKaRa, Japan). Ubiquitin (UBQ) was used as reference gene to calculate the relative levels of gene expression. The qPCR program was as follows: 95 °C for 30 s; 40 cycles of 95 °C for 5 s, 60 °C for 35 s; 95 °C for 1 min, 55 °C for 30 s, 95 °C for 30 s. All the primers used are listed in Table S8 . In addition, the genomic DNA for promoter cloning, transgenic and mutant identification was extracted by a CTAB method (Rogers and Bendich, 1985 (link)).
5
Epididymal Fat RNA Extraction and qPCR
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Total RNA from epididymal fat tissue was extracted with TRIzol Reagent (TaKaRa, Japan) and then reverse-transcribed to cDNA with a PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) kit (TaKaRa, Japan). Determinations of mRNA levels were performed with TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Japan) and a 7500 Fast Real-Time PCR system (ABI, USA). Thermal cycling consisted of an initial cycle of 95 °C for 5 minutes, followed by 40 amplification cycles of 95 °C for 15 s and 60 °C for 1 minute. The target genes were TLR4, IL6, IL1β, CD11c, IKKε and a housekeeping gene (Gapdh), and their primer sequences are listed in Supplementary Table S1 .
6
RT-qPCR Analysis of Gene Expression
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The same batch of samples was used for RT-qPCR detection. According to the manufacturer’s protocol, the total RNA of cells and lung tissue was extracted using RNAiso Plus reagent (Takara, Kyoto, Japan). The quality and concentration of RNA were detected using NanoDrop 2000 spectrophotometer (Seamer Fischer Technology Co., Ltd., Wilmington, TX, USA). One ug RNA was extracted from each sample for detection. Reverse transcription RNA was performed using PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (both purchased by Takara). The reverse transcription regimen lasted 15 min at 37 °C and 5 s at 85 °C. Then, the SYBR Green PCR kit (Takara, Kyoto, Japan) was used on a real-time PCR detection system (Quant-Studio 7 Flex, Thermo Scientific, Waltham, MA, USA). According to the actual manufacturer’s protocol, the thermal cycle condition is 95 °C for 30 s, followed by a 40 thermal cycle of 95 °C for 3 s and 60 °C for 30 s. The fold changes of gene expression were calculated using the 2−ΔΔCT method by normalizing β-Actin. Each analysis was repeated three times using different donors. The primers used were provided by Sangon Biotech (Shanghai, China). The primer sequence is shown in Table 1 .
7
Isolation and Sequencing of MaPepDA
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Genomic DNAs were isolated from mycelium using DNA Extraction Kit (OMEGA, Georgia, United States). Fungal total RNAs were extracted from mycelia using RNA Extraction Kit (CWBIO, Beijing, China). Complementary DNA (cDNA) synthesis was performed following the manufacturer’s instructions of the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) Kit (TaKaRa, Dalian, China). The whole cDNA sequence of MaPepDA was amplified using primers MaPepD-CF and MaPepD-CR (Supplementary Table S1 ). The pMD19-T vector was used for cDNA sequencing (TaKaRa, Dalian, China).
8
Anthocyanin and Sugar Metabolism Gene Expression
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Candidate gene sequences related to anthocyanin synthesis and sugar metabolism were searched fome Genbank. for in the ‘Sanyueli’ plum and Prunus domestica gene sequence libraries using the TBtools tool, and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi ) for comparison (details are shown in Table S1
Total RNA was extracted using the RNA prep Pure polysaccharide polyphenol plant total RNA (TIANGEN biotech, Beijing, China) and the Prime ScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (TaKara, Dalian, China) for reverse transcription. All steps were performed according to the reagent instructions. Quantitative PCR was conducted using the TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKara, Dalian, China). The reaction procedure was 95 ℃ for 30 s, 95 ℃ for 5 s, and TM for 30 s for 40 cycles. Each treatment was designed with three biological repetitions. The melting curve was analyzed, and the relative expression levels of each gene were calculated by the 2-ΔΔCt method.
Total RNA was extracted using the RNA prep Pure polysaccharide polyphenol plant total RNA (TIANGEN biotech, Beijing, China) and the Prime ScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (TaKara, Dalian, China) for reverse transcription. All steps were performed according to the reagent instructions. Quantitative PCR was conducted using the TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKara, Dalian, China). The reaction procedure was 95 ℃ for 30 s, 95 ℃ for 5 s, and TM for 30 s for 40 cycles. Each treatment was designed with three biological repetitions. The melting curve was analyzed, and the relative expression levels of each gene were calculated by the 2-ΔΔCt method.
9
Quantitative Analysis of Plant Immune Responses
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Total RNA was extracted from the leaves collected above. The first-strand cDNA was synthesized using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (TaKaRa, Dalian, China). Primers (Table S1 ) for GT1 were designed based on its sequence using Primer5 software. Primers for resistant genes PR1, RIN4 and ICS1 were as previously reported. The qRT-PCR reaction was performed using the SYBR® Premix Ex TaqTM (TaKaRa, China). Relative expression of the target genes was calculated using the 2−ΔΔCt method [62 (link)] with three technical replicates. Hydrogen peroxide was determined using a commercial kit according to the manufacturer’s instruction (Genepioneer Biotechnologise, Hong Kong, China). The superoxide anion production rate was measured by a luminol-based assay as described by Wang and Luo [63 ]. Data were analyzed by one-way ANOVA.
10
Quantitative Real-Time PCR Protocol
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qRT-PCR was performed as previously described (Wang et al., 2017 (link)). Total RNA was extracted at 4°C using Trizol RNA isolating reagent (Thermo Fisher Scientific, Waltham, MA, United States) according to the established procedures. Total RNA (1000 ng) was reversely transcribed using PrimeScriptTM RT reagent kit with gDNA Eraser (perfect real time) kit (Takara, Japan) for the synthesis of a single-stranded cDNA library according to the manufacturer’s introduction. Gene-specific mRNA analyses were performed using the standard protocol of SYBR Premix Ex TaqTM II (TliRnaseH plus) kit (Takara, Japan). Relative quantification was performed using the comparative threshold method (ΔΔCT) and Gapdh gene was used as a reference to normalize the specific gene mRNA expression. After amplification, each qPCR product was sequenced using electrophoresis to ensure the specificity. The primers used were listed in Table 1 .
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