RT-qPCR was performed to verify the transcriptome data of the selected DETs. Total RNA was extracted using a Plant RNA Kit (Omega Bio-Tek, Guangzhou, China). First-strand cDNA was synthesised with 1 μg of total RNA using the PrimeScript RT reagent Kit with gDNA Eraser (RR047A, Takara, Japan). For RT-qPCR, the TB Green TM Premix Ex Taq TM II kit (TaKaRa, Dalian, China) was used to produce 20 μL reactions consisting of 2 μL cDNA (50 ng/L), 0.5 μL forward primer (10 μmol/L), 0.5 μL reverse primer (10 μmol/L), 10 μL TB Green Premix Ex Taq II (2×) (RR820Q, Takara, Japan) and 7 μL ddH2O. For PCR, denaturation was conducted at 95°C for 30 s, followed by 40 cycles of the following conditions: denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and final extension step at 95°C for 15 s. RT-qPCR was performed using a CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). ScTubulin was used as the reference gene for normalisation (Li et al., 2015 (link)). Relative abundance levels were calculated using the 2-ΔΔCt method (Livak and Schmittgen, 2001 (link)), with three biological replicates and three technical replicates. Primers used for RT-qPCR were designed using the NCBI primer BLAST program and are listed in Supplementary Table S1
Tb green premix ex taq 2 2
Manufactured by Takara Bio
Sourced in Japan, China
TB Green Premix Ex Taq II (2×) is a ready-to-use PCR master mix that contains all the necessary components, including TB Green Dye, for real-time PCR amplification. It is designed to provide reliable and consistent results in quantitative PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Tb green premix ex taqtm 2 kit, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Cfx96 real time pcr system, by Bio-Rad (1 mentions)
Plant rna kit, by Omega Bio-Tek (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Taq pcr starmix, by GenStar (1 mentions)
Sodium dodecyl sulfate (sds), by Biosharp (1 mentions)
Mda test kit, by Nanjing Jiancheng (1 mentions)
E z n a mollusc dna kit, by Omega Bio-Tek (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
6 protocols using tb green premix ex taq 2 2
1
Verification of Transcriptome Data by RT-qPCR
2
Quantifying Viral Gene Expression
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As previously described, after treatment with different concentrations of inhibitors, viral gene expression was measured using Prime STAR Max DNA Polymerase amplified cDNA. qPCR was performed to assess the temporal expression of selected CyHV-2 genes. The qPCR reactions contained 12.5 µL of TB Green Premix Ex Taq II (2×) (Takara, Japan), 0.4 µM forward/reverse primers, and 200 ng cDNA. The thermal cycling program comprised: 95 ℃ for 30 s and then 39 cycles of 95 ℃ for 5 s and 59 ℃ for 30 min. The qPCR reactions were repeated three times. To construct a standard curve, serial decimal dilutions were performed using DNA extracted from the virus, and all results were normalized to the relevant actin expression levels. The normalization calculation was performed using the standard procedure for absolute quantification. Statistical significance was calculated using one-way analysis of variance (ANOVA). Values were considered as significant (*) if they had a p value of 0.01 to 0.05, very significant (**) if they had a p value of 0.001 to 0.01, and extremely significant (***) if they had of p value 0.0001 to 0.001.
3
Comprehensive Biochemical and Molecular Analysis
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SDS was purchased from Biosharp Company of China, and the purity was 99%. Total protein quantitation kit, SOD, CAT, and MDA test kits were purchased from Nanjing Jiancheng Company of China for determination of the activities of SOD, CAT and MDA content. The E.Z.N.A.® Mollusc DNA kit was the product of Omega Bio-Tek Company for extraction of genomic DNA, and 2 × Taq PCR StarMix was purchased from GenStar Company. Trizol reagent was purchased from Thermo Fisher Technology Co., Ltd. for extraction of RNA. Reverse Transcription kit and TB Green premix Ex Taq II (2×) were purchased from TaKaRa Company. The sequences of 13 random primers and qPCR primers used in this study are shown in Tables S1 and S2 .
4
Quantitative PCR for Gene Expression Analysis
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Using the BIORAD CFX96 Touch fluorescence quantitative PCR equipment, qPCR was carried out using β-actin as an internal standard after the mRNA was transformed to cDNA using a PrimeScriptTM RT reagent Kit (TaKaRa, China) (Bio-Rad Laboratories, CA, USA). The reaction system was composed of 10 L of ddH2O, 5 L of TB Green Premix Ex Taq II (2×)(Takara, China), 0.4 L of a particular forward/reverse primer, and 0.8 L of cDNA. The primers were listed in Table 1
5
Quantitative RT-PCR for Gene Expression
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The quantitative RT-PCR (qRT-PCR) reactions contained 10 µL TB Green premix Ex Taq II (2×) (TaKaRa), 0.8 µL PCR forward primer (10 µM), 0.8 µL PCR reverse primer (10 µM), 0.4 µL ROX reference dye or dye II (50×) (TaKaRa), 2 µL 1× reverse transcription reaction solution (cDNA solution from RT-PCR), and 6 µL sterile water. The hnRNP G primers were, forward primer 5′-AAA CTT TGG ACC ACA CAT ATC C-3′ and reverse primer 5′-AAG CCA CGC TTA CAC ATACTA-3′, and the internal reference β-actin primers were forward primer 5′-CCG TGA AAA GAT GCC CAG AT-3′ and reverse primer 5′-GGA CAG TGA GGCCAGGATAGA-3′.
The qRT-PCR reactions were run on a StepOne plus real-time PCR system (Thermo Fisher Scientific) using the following amplification conditions: pre-denaturation at 95°C for 30 seconds and 40 cycles at 95°C for 5 seconds and 60°C for 30 seconds. An amplification curve and a fusion curve were generated after the completion of qRT-PCR. GraphPad Prism software 9.3 was used for the analysis of these two curves (GraphPad Prism, Version X; San Diego, CA, USA,www.graphpad.com ). Gene expression was normalized using the 2–ΔΔCT method (Livak abd Schmittgen, 2001) following the GraphPad Prism instructions, with β-actin mRNA expression as an internal reference.
The qRT-PCR reactions were run on a StepOne plus real-time PCR system (Thermo Fisher Scientific) using the following amplification conditions: pre-denaturation at 95°C for 30 seconds and 40 cycles at 95°C for 5 seconds and 60°C for 30 seconds. An amplification curve and a fusion curve were generated after the completion of qRT-PCR. GraphPad Prism software 9.3 was used for the analysis of these two curves (GraphPad Prism, Version X; San Diego, CA, USA,
6
Quantitative Analysis of miRNAs and Genes
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RNA isolated was the same as “Preparation of RNA-seq Samples”, the efficiency of chi-miR-133a-3p mimics and inhibitor was detected by miRNAs qPCR. mRNA was reverse transcribed by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA), and the cDNA was used to detect the genes expression level. Ubiquitously expressed transcript gene (UXT) was selected as internal reference gene to normalize the mRNA levels in adipocyte. The qPCR system included TB Green Premix Ex Taq II (2×) (Takara, China) 10 μL, sense/antisense primer 1 μL, cDNA 1 μL, ddH2O up to 20 μL, 95°C 3 min, 95°C 10 s, melting-out temperature (TM) 10 s, 72°C 15 s, 40 cycles, primer information was shown as Table 2 .
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