Total RNA was extracted from mock- and MeJA-treated pomegranate leaves using the RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China). Reverse transcription (RT) was performed using total RNA and the PrimeScript™ RT Reagent Kit (Takara Bio Inc., Kusatsu, Japan). Quantitative PCR (qPCR) was carried out using the TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) kit (Takara) and a StepOnePlus Real-Time PCR System (ThermoFisher Scientific). Melting curve analysis was conducted and showed a single amplification product for each primer pair. For the RT-qPCR analysis, three biological replicates and each with three technical replicates were examined for mock- and MeJA-treated samples. Gene expression was analyzed using the comparative Ct (∆∆Ct) method (Livak and Schmittgen 2001 (link)) and significance levels were determined using a two-tailed Student’s t test. The primer sequences for the real-time qPCR analysis and the amplification efficiencies of the primer pairs are shown in Table S1.
Tb green premix ex taq tli rnaseh plus kit
Manufactured by Takara Bio
Sourced in Japan, United States, China
The TB Green® Premix Ex Taq™ (Tli RNaseH Plus) Kit is a ready-to-use reagent for quantitative real-time PCR (qPCR) analysis. It contains a proprietary Tli RNaseH Plus enzyme, TB Green® Dye, and other necessary components for sensitive and specific detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (6 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Rnaiso plus kit, by Takara Bio (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Takara primescript first strand cdna synthesis kit, by Takara Bio (1 mentions)
Quantstudio 7 flex real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Eco real time pcr system, by Illumina (1 mentions)
Primerscript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
Real time pcr detection system mastercycler, by Eppendorf (1 mentions)
Trizol, by Takara Bio (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
Minibest plant rna extraction kit, by Takara Bio (1 mentions)
32 protocols using tb green premix ex taq tli rnaseh plus kit
1
Quantitative RT-PCR Analysis of MeJA-Treated Pomegranate
2
Quantitative RT-PCR Analysis of Gene Expression
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RNA was reverse-transcribed using the PrimeScript RT reagent kit with gDNA Eraser (Takara Bio). The amplifications were carried out using TB Green Premix Ex Taq (Tli RNaseH Plus) kit (Takara Bio). qRT-PCR was performed on an ABI StepOnePlus real-time PCR system. Actin and beta-tubulin were used as endogenous controls. The relative gene expression levels were calculated using the 2–∆∆Ct method [63 (link)]. The sequences of the primers used for qRT-PCR are listed in Table S3 .
3
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from above-mentioned samples using the Tiangen RNAprep plant kit (Tiangen). We used RNase-free DNase1 to remove genomic DNA contamination before dissolving RNA (Tiangen). Additional PCR reactions and agarose gel electrophoresis were used to recheck the purity of RNA. Then, we used the Takara PrimeScript First-Strand cDNA Synthesis kit (Takara) to synthesize first-strand cDNA. All first-strand cDNA samples were diluted 5 times and stored at −20 °C for real-time quantitative PCR (qRT-PCR) experiments. All specific quantitative primers were designed by Primer Premier 5.0 and are shown in Table S2 . Real-time quantitative reverse transcription PCR was performed using the TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) kit (Takara) with the QuantStudio™ 7 Flex Real-Time PCR instrument (Applied Biosystems). A 20-µL reaction system was used, and each reaction contained 0.4 µL gene-specific primers, 0.4 µL ROX Reference Dye, 2 µL cDNA sample, 6.8 µL ddH2O and 10 µL TB Green Premix Ex Taq reagent. The relative expression level was evaluated based on the 2−ΔΔCT method, and tonoplast intrinsic protein 41 (TIP41) gene was used as the reference gene [73 (link)].
4
Wheat Gene and miRNA Expression Analysis
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The total RNA of all samples was extracted using TRIzol reagent (Invitrogen, CA, USA) following the manufactures’ instructions, and approximately 1 µg RNA was reverse transcribed with PrimerScript 1st Strand cDNA synthesis kit (TaKaRa, Shiga, Japan) and miRcute Plus miRNA cDNA T (Tingen, Beijing, China) for genes and miRNAs, respectively, according to the manufacturer protocol. qRT-RCR assays for TaGH9 and miRNAs were conducted on an Eco Real-time PCR system (Illumina, CA, USA) using TB Green Premix Ex Taq (Tli RNaseH Plus) kit (TaKaRa, Shiga, Japan) and SYBR® miRcute Plus miRNA PreMix (Tingen, Beijing, China), respectively, as described previously [28 (link),75 (link)]. Wheat 18S gene served as the reference gene for TaGH9 analysis, and U6 was used as a reference gene for miRNA analysis. The relative expression levels were calculated according to the 2–ΔΔCt method for relative expression with three biological replicates and three technical replicates [76 (link)]. The primers used in this study were designed using the Primer premier 5.0 (Primer, CAN, UK) program and are listed in Table S4 .
5
RNA Extraction, cDNA Synthesis, and qPCR Analysis
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Total RNA was extracted and purified with a RNAiso Plus kit (9109; TaKaRa bio-INC., Kusatsu, Shiga, Japan). cDNA was synthesized with a PrimeScript™ RT Master Mix Kit (RR036A; TaKaRa, Kusatsu, Shiga, Japan). Then, qPCR detection was conducted on an ABI Prism® 7500 real-time PCR detection system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) with a TB Green® Premix Ex Taq™ (Tli RNaseH Plus) kit (RR420A; TaKaRa, Kusatsu, Shiga, Japan). The transcriptional expressions of C/EBP homologous protein (CHOP) and heme oxygenase 1 (HO-1) were measured by qPCR. The primers used in the present study were as follows: HO-1, 5′-CCAGCGGGCCAGCAACAAAGTGC-3′, 5′-AAGCCTTCAGTGCCCACGGTAAGG-3′; CHOP, 5′-GACCTGCAAGAGGTCCTGTC-3′, 5′-TGTGACCTCTGCTGGTTCTG-3′; and GAPDH, 5′-TGACGCTGGGGCTGGCATTG-3′ and 5′-GGCTGGTGGTCCAGGGGTCT-3′. Relative gene expression was calculated with the 2−ΔΔCt method with GAPDH as a loading control. At least three independent samples were performed in each group, and each sample was measured in triplicate.
6
Quantitative Gene Expression Analysis of Fungal Spores
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For RNA isolation, 200μl of a 5×104conidia/ml spore suspension was spread onto cellophane membranes over CM plates and cultured for 3days. Total RNA was extracted with Trizol, following the manufacturer’s procedure (TaKaRa, Japan), and transcribed into cDNA using the PrimeScriptTM RT reagent kit with gDNA Eraser (TaKaRa, Japan). The qPCR assay was performed on the Real-Time PCR Detection System MasterCycler (Eppendorf, Germany) with the TB Green® Premix Ex Taq™ (Tli RNaseH Plus) kit (TaKaRa, Japan) following the manufacturer’s protocol. Relative abundance of transcripts was assessed by the 2−∆CT method, where ∆CT=CTgene−(CT40S+CTACTIN)/2. 2−∆∆CT was used as the standard for calculating fold changes between two strains, where ∆∆CT=∆CTstrain_1−∆CTstrain_2 (Livak and Schmittgen, 2001 (link)). Tukey’s HSD test was used to assess significance for all experimental data between samples (Tang and Zhang, 2013 (link)).
7
FOXA1 Gene Expression Analysis
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TRIzol reagent was used for the extraction of total cell RNA, and a reverse transcription reagent was used for cDNA synthesis. The TB Green™ Premix Ex Taq™TliRnaseH Plus kit (TaKaRa, Japan, RR420A) was used for PCR with GAPDH (upstream, 5′-TCGGAGTCAACGGATTTGGT-3′, and downstream, 5′-TTCCCGTTCTCAGCCTTGACGAPDH-3′) as an internal reference. The following primers for FOXA1 were used: upstream, 5′-CTACTACgCAGACACGCAGG-3′, and downstream, 5′-TCATGTTGCCGCTCGTAGTC-3′. The 20 μL reaction system was subjected to the following reaction conditions: predenaturation at 95°C for 30 s, 40 cycles of 95°C for 5 s and 60°C for 34 s, 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. Each experiment was replicated in three wells. The RT–qPCR results were analyzed by the 2−△△CT method. The experiment was repeated three times.
8
RNA Extraction and RT-qPCR Analysis
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Limbs for RNA isolation were stored in RNAlater (#AM7024, Invitrogen) at –20°C until all samples were collected. RNA isolation was performed using the RNAeasy Mini Kit. 50ng of RNA were used for cDNA synthesis using the PrimeScript RT reagent Kit (#RR037A, Takara, Göteborg, Sweden) following the provider’s instructions. RT-qPCR was performed using the TB Green Premix Ex Taq (Tli RNAseH Plus) kit (#RR420A, Takara). RT-qPCR was done using a LightCycler 480 system with a pre-defined protocol for SYBR Green. Results were analyzed using the ΔΔCT method and the Rpl4 housekeeping gene. After analysis, results were shown as relative levels compared to a control.
9
Quantitative RT-PCR Analysis of Plant Gene Expression
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Total RNA was isolated from the leaves using the MiniBEST Plant RNA Extraction Kit (9769, TaKaRa, Japan). Subsequently, first-strand cDNA synthesis was performed using the PrimeScript™ RT Master Mix (RR036A, TaKaRa, Japan). A PCR reaction mixture (20 μL) was prepared according to the manual of the TB Green® Premix Ex Taq™ (Tli RNaseH Plus) Kit (RR820A, TaKaRa, Japan), in accordance with the manufacturer’s instructions. The reaction was conducted using the BIO-RAD CFX Connect™ Real-time System (BIORAD, USA). The DoACTIN gene was employed as an internal control [62 (link)]. Each experiment was replicated in triplicate, and three biological replicates were conducted. The primers used in the qRT-PCR experiments are listed in Table S3. Relative expression levels were calculated according to the Normalized Expression method (2−△△CT method) [63 (link)].
10
Transcriptional Analysis of MRSA Biofilm Genes
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After incubation of MRSA suspensions with TPCP (1× MIC) for 8 and 16 h, total RNA was extracted by using the Trizol method (Ambion, USA). A control group was set up separately. Total RNA was reverse-transcribed into cDNA at approximately 1 μg RNA concentration using RevertAid Master Mix and DNase I (Thermo Fisher Scientific, USA). The expressions of biofilm-related (icaA, agrA, sarA, and sigB) and eDNA-related genes (cidA) were tested by RT-PCR using TB Green Premix Ex Taq (Tli RNaseH Plus) Kit (Takara, Japan). gyrB was used as the reference gene and the results were analyzed by 2-△△Ct method. The experiment was repeated three times. The primers used are listed in Table 1 .
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