Expression levels of chitinase-like genes in B. tabaci were determined by qRT-PCR with gene-specific primers designed by Primer premier 5.0 (Table S1 ). ABI PRISM 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) was used for conduction of qRT-PCR with a 20-μL reaction system containing 0.4 μL of 50 × ROX reference dye (TIANGEN, Beijing, China), 0.6 μL of each specific primer, 1 μL of cDNA template, 7.4 μL of ddH2O, and 10 μL of 2 × SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China). The qRT-PCR program was as follows: 95 °C for 10 min (initial denaturation), followed by 40 cycles of 95 °C for 5 s (denaturation), 60 °C for 15 s (annealing), and 72 °C for 35 s (elongation). qRT-PCR primers which meet the amplification efficiencies of 90%–110% were used and listed in Table S1 . Relative expression levels were quantified using the 2−ΔΔCt method [49 (link)]. Two reference genes 60S ribosomal protein L29 (RPL29) (GenBank accession no. EE596314) and elongation factor 1 alpha (EF1-α) (GenBank accession no. EE600682) were used for normalization of target genes expression [50 (link)]. For each sample, three biological replicates and four technical replicates were performed.
Superreal premix plus sybr green
Manufactured by Tiangen Biotech
Sourced in China, United States, Switzerland
SuperReal PreMix Plus (SYBR Green) is a ready-to-use solution for real-time PCR (qPCR) experiments. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, and all the necessary components for efficient and sensitive DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (46 mentions)
Fastquant rt kit, by Tiangen Biotech (31 mentions)
Rnasimple total rna kit, by Tiangen Biotech (21 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (21 mentions)
Fastking rt kit with gdnase, by Tiangen Biotech (20 mentions)
Fastking rt kit, by Tiangen Biotech (16 mentions)
Fastquant rt kit with gdnase, by Tiangen Biotech (16 mentions)
Trizol, by Thermo Fisher Scientific (13 mentions)
Trizol reagent, by Tiangen Biotech (12 mentions)
Fastking gdna dispelling rt supermix, by Tiangen Biotech (10 mentions)
Dnase 1, by Thermo Fisher Scientific (10 mentions)
Cfx96 real time system, by Bio-Rad (7 mentions)
Primescript rt reagent kit, by Takara Bio (7 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (7 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (6 mentions)
Formic acid, by Thermo Fisher Scientific (6 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (6 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Cfx96 real time pcr system, by Bio-Rad (5 mentions)
258 protocols using superreal premix plus sybr green
1
Quantifying Chitinase-like Genes in Bemisia tabaci
2
qRT-PCR Analysis of COEs in B. tabaci
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The specific primers for COEs of B. tabaci MED were designed using Primer Premier 5.0 and used to verify the expression levels of COEs by qRT-PCR (Table S2 ). qRT-PCR was performed using an ABI 7500 system (Applied Biosystems) with the 25-μL reaction containing 0.5 μL of each specific primer, 0.5 μL of 50 × ROX reference dye (TIANGEN, Beijing, China), 1 μL of cDNA template, 10 μL of ddH2O, and 12.5 μL of 2 × SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China). The qRT-PCR programme was as follows: 95 °C for 10 min (initial denaturation), followed by 40 cycles of 95 °C for 5 s (denaturation), 60 °C for 15 s (annealing), and 72 °C for 35 s (elongation). Only the qRT-PCR primers with 90–110% amplification efficiencies were used for the subsequent data analysis.
Relative expression levels were quantified using the 2−ΔΔCt method [66 (link)]. The geometric mean of the reference genes 60S ribosomal protein L29 (RPL29) (GenBank accession no. EE596314) and elongation factor 1 alpha (EF1-α) (GenBank accession no. EE600682) was used to normalize the expression of target genes [67 (link),68 (link)]. Three biological replicates and four technical replicates were performed for each sample. One-way analysis of variance (ANOVA) (SPSS 23) was used to detect significant differences between samples.
Relative expression levels were quantified using the 2−ΔΔCt method [66 (link)]. The geometric mean of the reference genes 60S ribosomal protein L29 (RPL29) (GenBank accession no. EE596314) and elongation factor 1 alpha (EF1-α) (GenBank accession no. EE600682) was used to normalize the expression of target genes [67 (link),68 (link)]. Three biological replicates and four technical replicates were performed for each sample. One-way analysis of variance (ANOVA) (SPSS 23) was used to detect significant differences between samples.
3
Quantification of Gene Expression by RT-qPCR
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Total RNA was extracted using TRIzol Reagent (T9424, Sigma Aldrich, St Louis, MO, USA) and reverse transcribed into cDNA using Fastking RT kit (KR116-02, Tiangen, Beijing, China) according to the manufacturer’s instructions. The specific quantitative primers are listed in Supplementary Table S4 . A set of cDNA, 2× SuperReal PreMix Plus SYBR Green (FP205, Tiangen, Beijing, China) and forward and reverse primers were mixed and subjected to RT-qPCR performed on CFX96 Touch Deep Well Real-Time PCR Detection System (CFX96, Bio-rad, Hercules, CA, USA). The program was as follows: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 30 s. The reaction was performed in a CFX96™ Real-Time PCR machine (CFX96, Bio-rad, Hercules, CA, USA). The standard curve was used to determine PCR efficiency. The dissociation curve of amplified products was used to evaluate product specificity. Relative expression levels were normalized to β-Actin using the 2−ΔΔCt method. For each gene, three technical replicates and three biological replicates were assayed.
4
Quantitative Gene Expression Analysis in Mouse Kidney
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Total RNA was extracted from mouse kidney using TRIzol reagent (BioTake Corporation) according to the manufacturer's instruction. Approximately 500 ng total RNA from each sample was used for reverse transcription and cDNA synthesis was performed using TIANGEN reverse transcription kit. PCR primers were designed and synthesized from TSINGKE Biological Technology. cDNA (1 μl/sample) was used for qPCR on a Roche Light Cycler 480. Reactions were performed in a 20 μl reaction mixture containing 10 μl of the 2× SuperReal Premix Plus (SYBR Green, TIANGEN BIOTECH). The sequences of the primers used for real‐time PCR are listed in Table S1 . Relative changes in gene expression were calculated using 2−ΔΔCT, and all experiments were repeated at least three times.
5
Quantitative Analysis of BtFAD2 Expression
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The expression levels of target genes were quantified using the QuantStudio 3 Real‐Time PCR System (Applied Biosystems). The gene‐specific primers of BtFAD2 used for the real‐time quantitative PCR (qPCR) analysis were designed by the Primer Premier 5.0 software (Table S1 , Supporting Information). The 25 µL PCR reactions included 0.5 µL of 50 × ROX Reference Dye (TIANGEN), 0.75 µL of each specific primer, 1 µL of cDNA template, 9.5 µL of ddH2O, and 12.5 µL of 2 × SuperReal PreMix Plus (SYBR Green) (TIANGEN). The qPCR reactions were performed in an ABI 7500 system (Applied Biosystems) with the following protocol: initial denaturation of 94 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The amplification efficiencies were determined by dissociation curve analysis using five two‐fold serial dilutions of B. tabaci cDNA template. Only primers with 90%–110% amplification efficiencies were used for the subsequent studies.
Relative quantification was calculated according to the 2−ΔΔCt method,[47 (link)
] to accurately analyze the expression of the target genes, the expression data were normalized to the internal gene elongation factor 1 alpha (EF1‐a) (GenBank accession number EE600682). Three independent biological replicates and four technical replicates were performed for each whitefly sample.
Relative quantification was calculated according to the 2−ΔΔCt method,[47 (link)
] to accurately analyze the expression of the target genes, the expression data were normalized to the internal gene elongation factor 1 alpha (EF1‐a) (GenBank accession number EE600682). Three independent biological replicates and four technical replicates were performed for each whitefly sample.
6
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated using the MiniBEST Universal RNA Extraction Kit (TAKARA, Japan) according to the manufacturer's protocol. The quality and quantity of the isolated RNA were analyzed on Tgem Pro spectrophotometer (Tiangen Biotech, China). After genomic DNA elimination, cDNA was synthesized from 50 ng total RNA using FastKing One-step Synthesis Premixed Kit according to the manufacturer's protocol (Tiangong Biotech, China). Quantitative real-time PCR was performed with the use of 2×SuperReal PreMix Plus (SYBR Green, Tiangen Biotech, China) on the A600 Super Gradient Cycler (LonGene, China) following the official protocol. The primer sequences are presented in the Additional file: Table S1. Data were analyzed with GraphPad Prism 7.
7
Analyzing Chondrocyte Gene Expression
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SW1353 cells (5x105 in each dish) were seeded in a 6 cm dish and treated with 10 ng/ml IL-1β and/or 20 mM irisin at 37˚C for 24 h. The cells were harvested and then washed with PBS. Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Then, 5 µl RNA was used for electrophoresis with a 1% agarose gel to detect the integrity of the RNA. The cDNA was synthesized from 1 µg total RNA using cDNA synthesis (Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions. The expression levels of MMP-13, Col II and Wnt-1 were analyzed using the primer sequences and β-catenin as the reference gene as listed in Table I .
The experimental operation of the reaction system was carried out according to the manufacturer's instructions. The 20 µl reaction mixture consisted of 10 µl 2*SuperReal PreMix Plus (SYBR Green; Tiangen Biotech Co., Ltd.) and each primer. The StepOne System (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for qPCR using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The thermocycling conditions were as follows: Initial denaturation at 95˚C for 1 min; 40 cycles of 95˚C for 10 sec, 58˚C for 30 sec and 72˚C for 30 sec; and then the melt curve was analyzed. Target gene levels were analyzed using the 2-ΔΔCq method (14 (link)).
The experimental operation of the reaction system was carried out according to the manufacturer's instructions. The 20 µl reaction mixture consisted of 10 µl 2*SuperReal PreMix Plus (SYBR Green; Tiangen Biotech Co., Ltd.) and each primer. The StepOne System (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for qPCR using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The thermocycling conditions were as follows: Initial denaturation at 95˚C for 1 min; 40 cycles of 95˚C for 10 sec, 58˚C for 30 sec and 72˚C for 30 sec; and then the melt curve was analyzed. Target gene levels were analyzed using the 2-ΔΔCq method (14 (link)).
8
Notch Signaling and Osteogenesis in Ligamentum Flavum
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The expression levels of Notch signaling pathway genes and osteogenic markers genes in ligamentum flavum cells were assessed by real-time RT-PCR at the indicated time points. Total RNA was isolated from cells using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (K1622; Thermo). SYBR green-based quantitative real-time PCR (qPCR) was performed in 96-well plates using Super-Real PreMix Plus SYBR Green (FP205; TIANGEN, Beijing, China), and an iCycler Multicolor Real-Time PCR Detection system (BIO-RAD, Hercules, VA). Values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the 2 ÀDCt method or corresponding groups using the 2 ÀDDCt method. Table 2 describes the detailed primer sequences.
9
Differential Expression of Esophageal Cancer Genes
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Six genes (RP11‐25G10.2, ADAMTS9‐AS1, ZBED3‐AS1, SFN, ATP1A2, and GNA15) were selected as candidate genes. Eighteen tissue samples were obtained, including nine samples of ESCC para‐cancer, two tumor samples of grade 3 ESCC, and seven tumor samples of grade 2 ESCC. This study was approved by the ethics institute of our hospital. The signed informed consent was obtained from all the participants.
Total RNA was isolated using a TRizol kit (Tiangen, China). A Fast Quant RT Kit (Tiangen, China) was utilized to obtain the complementary DNA. Using Super Real PreMix Plus SYBR Green (Tiangen, China), quantitative real‐time PCR was generated using the LightCycler 96. Relative gene expression was analyzed by the 2−ΔΔCt method.
Total RNA was isolated using a TRizol kit (Tiangen, China). A Fast Quant RT Kit (Tiangen, China) was utilized to obtain the complementary DNA. Using Super Real PreMix Plus SYBR Green (Tiangen, China), quantitative real‐time PCR was generated using the LightCycler 96. Relative gene expression was analyzed by the 2−ΔΔCt method.
10
Quantitative Gene Expression Analysis Protocol
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Total RNAs were extracted from each seedling root of the treatment and control groups separately, according to the manufacturer’s protocol for the RNAsimple Total RNA Kit (Tiangen, Shanghai, China). Extracted RNA was reverse-transcribed into cDNA using FastKing cDNA (Tiangen, Shanghai, China). Specific PCR primers of the eight selected genes were designed using Primer5 (Table S2 ). Samples and standards were performed on a QuantStudio 5 Real-Time PCR System (Thermo Scientific, Massachusetts, USA) using the SuperReal PreMix Plus (SYBR Green) (Tiangen, Shanghai, China). Three biological replicates were included. Each Real-Time PCR was performed in a 20 µL reaction volume containing 10 µL SuperReal PreMix Plus, 6 µL ddH2O, 0.8 µL forward primer (10 µmol/L), 0.8 µL reverse primer (10 µmol/L), 0.4 µL ROX Reference Dye and 2 µL template cDNA. The PCR programs were run as follows: 10 min of pre-denaturation at 95 °C, 40 cycles of 15 s at 95 °C, 30 s at 60 °C, melt curve stage: 15 s at 95 °C, 1 min at 60 °C, and 15s at 95 °C. The transcript abundance of selected genes was calculated by 2−ΔΔCt method and normalized with the results of the 18s gene [96 (link)]. The primers for RT-PCR are shown in Supplemental Table S9 .
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