After optimization of the reaction parameters, both the singular qPCR and duplex real-time qPCR assays were performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). For the singular qPCR assay for FPV, the reaction volume was 20 μL, comprising 0.6 μL each of forward and reverse primers, 10 μL of 2 × SuperReal Premix Plus (Tiangen), 1 μL of DNA template, and RNase-free H2O (to make up final volume). The singular qPCR assay for FBoV-1 had the same reaction components as the reaction for FPV. The reaction volume for the duplex real-time qPCR assay was 40 μL, containing 0.2 μL of each primer for FBoV-1, 0.8 μL of each primer for FPV, 1 μL of FPV DNA template, 1 μL of FBoV-1 DNA template, and 20 μL of 2 × SuperReal Premix Plus (Tiangen). RNase-free H2O was added to make up final volume. The reaction conditions for both singular and duplex qPCR were as follows: 15 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. The annealing/extension temperature was then optimized. The reaction without template and the negative reference samples were used as negative controls.
Superreal premix plus
Manufactured by Tiangen Biotech
Sourced in China, United States, Germany, Canada
SuperReal PreMix Plus is a qPCR (quantitative Polymerase Chain Reaction) reagent designed for real-time PCR applications. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, pre-mixed and ready-to-use. The product is intended to simplify and streamline the qPCR setup process.
Automatically generated - may contain errors
Lab products found in correlation
Fastquant rt kit, by Tiangen Biotech (41 mentions)
Trizol reagent, by Thermo Fisher Scientific (39 mentions)
Fastking rt kit, by Tiangen Biotech (17 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (16 mentions)
Fastquant rt kit with gdnase, by Tiangen Biotech (14 mentions)
Rnasimple total rna kit, by Tiangen Biotech (14 mentions)
Trizol, by Thermo Fisher Scientific (12 mentions)
Trizol reagent, by Tiangen Biotech (12 mentions)
Fastking gdna dispelling rt supermix, by Tiangen Biotech (11 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (8 mentions)
Mircute plus mirna qpcr kit, by Tiangen Biotech (7 mentions)
Rnaprep pure cell bacteria kit, by Tiangen Biotech (6 mentions)
Fastking rt kit with gdnase, by Tiangen Biotech (6 mentions)
Mircute plus mirna first strand cdna kit, by Tiangen Biotech (6 mentions)
Mastercycler ep realplex, by Eppendorf (5 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (5 mentions)
Quantscript rt kit, by Tiangen Biotech (5 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (5 mentions)
Tianscript rt kit, by Tiangen Biotech (4 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (4 mentions)
282 protocols using superreal premix plus
1
Singleplex and Duplex qPCR Assays for FPV and FBoV-1
2
Quantification of miRNA and Smad7 Expression
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Total RNA was extracted with TRIZOL (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. 2μg RNA was reverse-transcribed to cDNA by stem-loop methods with the TIANScript RT Kit (Tiangen, Beijing, China). SuperReal PreMix Plus (SYBR Green) (Tiangen, Beijing, China) was used to detect the expression of the microRNAs, and the 5S was used as the internal reference. The stem-loop, forward primers, and the universal reverse primer were designed based on the sequences of rat miR-17, miR-181a, miR-34a, and 5S. The primer sequences are shown in Table 1 . For RT-qPCR analysis of Smad7, the total RNA was reverse-transcribed to cDNA using the TIANScript RT Kit (Tiangen, China). The PCR assay was implemented using SuperReal PreMix Plus (SYBR Green) (Tiangen, China), and GAPDH was used as the endogenous control gene. The primer sequences of Smad7 were as follows: forward, 5'-GGAGTCCTTTCCTCTCTC-3'; reverse, 5'-GGCTCAATGAGCATGCTTCAC-3'. The GAPDH primer sequences were as follows: forward, 5'-TCTCTGCTCCTCCCTGTTC-3'; reverse, 5'-ACACCGACCTTCACCATCT-3'. The primers were synthesized by Generay Company (Shanghai, China). PCR was performed on ABI7500 Real-Time PCR appliance. The cycling conditions were as follows: 95°C for 15 min, 95°C for 10 sec, and 60°C for 32 sec. The relative quantitative analysis of the expression was calculated using the 2−ΔΔCt method.
3
Quantitative PCR Analysis of Gene Expression
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Resulted cDNA above was used as template for qPCR. Primer Premier 5.0 software was used to designed primer pairs (Supplementary Table S2 ), and the parameters were set as 20bp±1bp in length, melting temperature (Tm) of 50–60°C and a product size of 150–250 bp (Priya et al., 2012 (link)). 20µl PCR system were mixed following the SuperReal PreMix Plus (Tiangen, Beijing, China) introductions, contained 10 µl 2×SuperReal PreMix Plus, 0.6 µl of each primer (10 mM), 1 µl cDNA and 7.8 µl RNase-free ddH2O. Reaction protocols were one cycle at 95°C (3 min), followed by 40 cycles of denaturation at 95 C (5s), annealing and extension at 60 C for 30 s, running in CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). A melting curve was generated from 65 to 95°C after each qPCR reaction, confirming the single peak and there was no primer-dimer or non-specific product. The TEF-1α (Priya et al., 2012 (link)) was selected as the internal control and the relative expression level of each gene was calculated by the 2−ΔΔCT method. Each sample had four biological replicates and three technical replicates.
4
Quantifying Whitefly Gene Expression
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Gene-specific primers for the AATs of B. tabaci B were designed with Primer Premier 5.0 and used in RT-qPCR analysis. The PCR reactions (total 25 μl) contained 12.5 μl of 2×SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China), 9.5 μl of ddH2O, 1 μl of cDNA template, 0.75 μl of each specific primer, and 0.5 μl of 50×ROX Reference Dye (TIANGEN, Beijing, China). The RT-qPCR were conducted with an ABI 7500 system (Applied Biosystems) with the following parameters: one cycles of 95°C for 3 min; 40 cycles of 95°C for 15 s, 60°C for 30 s, 72°C for 30 s. Five 2-fold serial dilutions of whitefly cDNA template were used to determine the RT-qPCR primers amplification efficiencies by dissociation curve analysis 50 (link). Only primers with 90-110% amplification efficiencies were used for the subsequent researches.
Relative quantification was calculated by the 2-ΔΔCt method 51 (link), and data were normalized to EF1-α (GenBank: EE600682) reference gene 52 (link). Three independent biological replicates and four technical replicates were used for each sample. Significant differences between samples were determined by one-way ANOVA with Holm-Sidak's tests (overall significance level = 0.05).
Relative quantification was calculated by the 2-ΔΔCt method 51 (link), and data were normalized to EF1-α (GenBank: EE600682) reference gene 52 (link). Three independent biological replicates and four technical replicates were used for each sample. Significant differences between samples were determined by one-way ANOVA with Holm-Sidak's tests (overall significance level = 0.05).
5
Quantifying BYDV-GPV Gene Expression in R. padi
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Total RNAs were extracted using TRIzol reagent (Invitrogen, California, USA) from four groups of R. padi: collected after 0 h, 12 h, 24 h, or 48 h acquisition access period [AAP] on BYDV-GPV-infected oat plants). First-strand cDNA of each sample was synthesized using the FastQuant RT Kit (Tiangen, Beijing, China) and the kit protocol. Actin was used as the internal reference gene. The obtained cDNAs were used as the template for the q-PCR. The q-PCR reaction mixture comprised 6.4 μL RNase-free ddH2O, 10 μL 2× SuperReal PreMix Plus (SuperReal PreMix Plus kit [SYBR Green I, Tiangen]), 0.6 μL forward primer (10 mM), 0.6 μL reverse primer (10 mM), 0.4 μL 50× ROX Reference Dye, 2 μL diluted cDNA (1:30 dilution of cDNA sample template) or ddH2O (as the no-template control). The reaction program were as follows: 95 °C, 15 min, 1 cycle; followed by 40 cycles of 95 °C 10 s, 59.2 °C 32 s, 60 °C 32 s. Relative gene expression was calculated according to the Livak method (2-ΔΔCt). The experiments were repeated 3 times independently. Primers used in RT-qPCR for validation of differentially expressed genes are shown in Supplementary Table S1 .
6
Quantifying StCDPKs mRNA Levels
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To investigate relative mRNA levels of StCDPKs, cDNA generated from the whole RNA in plant leaves was used for the qRT-PCR analysis. A reaction mixture for qPCR consisted of 100 ng of cDNA, 0.6 μL of specific primers (10 μM), 10 μL of 2× SuperReal PreMix Plus and 0.4 μL of 50× ROX Reference Dye (Tiangen Biotech, Beijing, China) to a final volume of 20 μL. The thermal profile of the ABI3000 system (Applied Biosystems, Foster City, CA, USA) were as follows: initial denaturation for 2 min at 94 °C, and 40 cycles of denaturation for 30 s at 94 °C, annealing for 34 s at 60 °C and extension for 30 s at 72 °C. The cycle threshold (CT) values were obtained and the mRNA level was calculated based on formula 2−ΔΔCt. Each experiment was carried out in three technical and three biological replicates. StEf1a was used as an internal control. Primer sequences were referenced from our previous study [34 (link)].
7
Quantitative PCR Analysis of Gene Expression
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We extracted total RNA from harvested cells by TRIzol reagent (Tiangen, Beijing, China) strictly following the manufacturer’s protocol and then used the NanoDrop ND-2000 spectrophotometer to measure the consistency and OD value to ensure the RNA quality. According to the concentration we measured, we obtained cDNA through the FastQuant RT Kit (with gDNase) following the manufacturer’s instructions (Tiangen, China). Then, we performed a q-PCR reaction with a 20 μL volume on a Mastercycler ep realplex2 system (Eppendorf, Germany). The q-PCR reaction mix consisted of 1 μL cDNA, 10 μL of 2×SuperReal PreMix Plus (Tiangen), and 0.5 μL of each primer, which was 10 μM. Water was added to a volume of 20 μL. The mRNA and miRNA primers are listed in the S1 File .
8
Quantitative Real-Time PCR Analysis of Cucumber
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Quantitative real-time PCR was conducted using the SYBR Green I 96-I system (Roche fluorescence quantitative PCR instrument, Basle). Reaction mixtures consisted of 4.5 μL of 2×SuperReal PreMix Plus (TianGen Biotech, China), a mixture of primers (0.2 μL of forward and reverse primer for proper gene), 4.3 μL of RNase-Free ddH2O and 1 μL of cDNA. The PCR program was set up in seven stages: (1) 95°C for 15 min (pre-incubation), (2) 95°C for 10 s, (3) 58°C for 20 s, (4) 72°C for 30 s, (3) repeated 40 times (amplification), (5) 95°C for 0.5 s, (6) 60°C for 1 min and (melt) (7) 50°C for 30 s (cooling). The primers were synthesized by BGI Tech (China), and the PCR reaction quality was estimated based on melting curves. 18s rRNA was used as an internal control for determining transcript levels in cucumber. The gene-specific primers employed are shown in S1 Table . Two independent biological replicates and three technical replicates for each biological replicate were run, and the significance was determined by t-test using SPSS statistical software (P < 0.05).
9
Quantitative Real-Time PCR for Gene Expression
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3T3-L1 and C2C12 cells were washed once in 1× phosphate-buffered saline (PBS) and total RNA was extracted using TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA). First-strand cDNA was synthesized from 1 μg of total RNA using a fast reverse transcription kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. Each 20 μL qRT-PCR mixed system included 8 μL of deionized water, 10 μL of 2× SuperReal PreMix Plus (TIANGEN), 1 μL of cDNA, and 0.5 μL each of forward and reverse primers (10 μM). The qRT-PCR conditions included denaturation at 95 °C for 180 s and 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. Gene expression was quantified using the Mastercycler ep realplex (Eppendorf, Hamburg, Germany) and 2−ΔΔCT method with GAPDH as the standard. The primer sequences are shown in Supplementary Table S2 .
10
Quantifying Copy Number of Lipase Genes
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The copy number of target genes in transformants exhibiting higher lipase activity was determined via qPCR on the DNA Engine Opticon Monitor 2 with the MJ-Opticon 2 system (MJ Research, MA, USA). According to the product manual of SYBR Green dye (Tiangen Biotech Co., Beijing, China), qPCRs were performed in a 20 μL mixture consisting of 7.8 μL of Rnase-free water, 0.6 μL of primer pairs (10 μM), 1 μL of template, and 10 μL of 2× SuperReal PreMix Plus (Tiangen Biotech Co., Beijing, China). The amplification reactions were initiated with 15 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 20 s at 58 °C, and 30 s at 72 °C. Then, the specificity of the amplification process was analyzed by constructing melting curves. The glyceraldehyde-3-phosphate dehydrogenase gene (Genbank accession no. U62648.1, GAP) of P. pastoris was set as the endogenous reference gene and cloned by the primer pair QGAP-F/QGAP-R. Tenfold dilution series of linearized vectors harboring reference or target genes were utilized as templates to build the standard curves. The copy number of the target genes was analyzed in terms of a previously described method [47 (link)]. All samples were determined in triplicate.
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