RT-qPCR analysis was performed according to standard procedures [43 (link)]. E. coli E. coli
Thunderbird sybr qpcr mix
Manufactured by Toyobo
Sourced in Japan, United States, Switzerland, China, Germany, Canada, Singapore, Australia
THUNDERBIRD SYBR qPCR Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) analysis using SYBR Green detection chemistry. It contains all the necessary components, including a hot-start DNA polymerase, for efficient and specific amplification of DNA targets.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (326 mentions)
Revertra ace qpcr rt master mix, by Toyobo (251 mentions)
Revertra ace qpcr rt kit, by Toyobo (171 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (166 mentions)
Rneasy mini kit, by Qiagen (141 mentions)
Revertra ace qpcr rt master mix with gdna remover, by Toyobo (111 mentions)
Revertra ace, by Toyobo (106 mentions)
Trizol, by Thermo Fisher Scientific (96 mentions)
Gdna remover, by Toyobo (72 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (53 mentions)
Rnaiso plus, by Takara Bio (51 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (48 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (46 mentions)
Steponeplus, by Thermo Fisher Scientific (45 mentions)
Lightcycler 96, by Roche (38 mentions)
Isogen, by Nippon Gene (37 mentions)
Isogen 2, by Nippon Gene (35 mentions)
Primescript rt reagent kit, by Takara Bio (35 mentions)
Thermal cycler dice real time system, by Takara Bio (34 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (33 mentions)
1 608 protocols using thunderbird sybr qpcr mix
1
RT-qPCR Analysis of E. coli Gene Expression
2
Quantitative RT-PCR Analysis of E. coli
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RT-qPCR was performed according to standard procedures [19 (link)]. E. coli cells were inoculated into M9 minimal medium supplemented with CAA (0.2%) at 37 °C with aeration by constant shaking at 150 rpm. Total RNA was extracted from exponential phase E. coli cells (OD600 = 0.4) using ISOGEN solution (Nippon Gene, Tokyo, Japan). Total RNA (1 μg) was transcribed into cDNA with random primers using the THUNDERBIRD™ SYBR® qPCR/RT Set (TOYOBO, Osaka, Japan). Quantitative P CR (qPCR) was performed using the THUNDERBIRD™ SYBR® qPCR Mix (TOYOBO) and a LightCycler® 96 system (Roche, Basel, Switzerland). The primer pairs are listed in Additional file 2 : Table S1A. The cDNA templates were serially diluted four-fold and used for qPCR analysis. The qPCR mixtures, containing 10 μL of THUNDERBIRD™ SYBR® qPCR Mix (TOYOBO), 1 μL of each primer (5 μM stock), 7 μL of water, and 1 μL of cDNA, were amplified under the following thermal cycling conditions: 2 min at 95 °C, 45 cycles of 10 s at 95 °C and 20 s at 55 °C, and then 20 s at 72 °C. The 16S rRNA expression level was used to normalize the phaC mRNA levels of the test samples, and the relative expression levels were quantified using Relative Quantification Software provided by Roche. The results are presented as the averages of three independent experiments.
3
Quantitative Gene Expression Analysis via RT-qPCR
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RT-qPCR analysis was performed according to the standard procedure (Shimada et al., 2015b (link)). E. coli cells were inoculated in LB medium at 37°C under aeration with constant shaking at 150 rpm until OD600 reached 0.3 or 1.5, following which total RNA was extracted. The total RNA was transcribed to cDNA using random primers and THUNDERBIRD SYBR qPCR RT set (TOYOBO, Osaka, Japan). Quantitative PCR (qPCR) was conducted using THUNDERBIRD SYBR qPCR mix (TOYOBO) and was performed using the LightCycler 480 system (Roche). The pairs of primers used in the experiment are described in Supplementary Table 1C . The cDNA templates were serially diluted fourfold and used in the qPCR assays. The qPCR mixtures, each containing 10 μL of THUNDERBIRD SYBR qPCR mix (TOYOBO), 1 μL of each primer (5 μM stock), 7 μL of water, and 1 μL of cDNA, were amplified under the following thermal cycling conditions: 95°C treatment for 2 min; 45 cycles of 10 s at 95°C and 20 s at 55°C; and incubation for 20 s at 72°C. The expression levels of 16S rRNA were used to normalize the RNA levels of test samples, and the relative expression levels were quantified using Relative Quantification software provided by Roche. The results presented are the averages of the results from three experiments.
4
RT-qPCR Analysis of E. coli Transcripts
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RT-qPCR analysis was performed according to standard procedures [57 (link)]. E. coli
5
Quantitative PCR Analysis of E. coli Transcripts
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RT-qPCR was performed according to standard procedures [35 (link)]. E. coli cells were inoculated into KM supplemented with glucose (0.25% or 1%) at 37 °C under aeration with constant shaking at 150 rpm. Total RNA was extracted from exponential phase E. coli cells (OD600 = 0.5) using ISOGEN solution (Nippon Gene, Tokyo, Japan). Total RNA was transcribed to cDNA using the THUNDERBIRD SYBR qPCR RT Set (TOYOBO, Osaka, Japan). Quantitative PCR (qPCR) was conducted using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) and a LightCycler 96 system (Roche). The primer pairs are listed in Table S1c . The cDNA templates were serially diluted four-fold and used for qPCR analysis. The qPCR mixtures, containing 10 μL of THUNDERBIRD SYBR qPCR Mix (TOYOBO), 1 μL of each primer (5 μM stock), 7 μL water, and 1 μL cDNA, were amplified under the following thermal cycling conditions: 95 °C treatment for 2 min, 45 cycles of 10 s at 95 °C and 20 s at 55 °C, and then incubated for 20 s at 72 °C. The 16S rRNA expression level was used to normalize the varying levels of the test samples, and the relative expression levels were quantified using Relative Quantification software provided by Roche. The results are presented as the average of three independent experiments.
6
Characterization of RNS1 Protein-DNA Interactions
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EMSA (electrophoretic mobility shift assay assay) was conducted as previously described [26 (link)]. The expression and preparation of the protein RNS1-DBD (the truncated RNS1 protein contain DNA binding domain, Glu-61 to Pro-267) in the E. coli strain BL21 (DE3) were also conducted as previously described [28 (link)]. The biotin-labeled and unlabeled DNA probes were commercially synthesized (TsingKe Biological Technology, Hangzhou, China). The sequences of the DNA probes were presented in Table S1 . The EMSA assays were conducted using Light Shift Chemiluminescent EMSA kit (Thermo Fisher, Waltham, MA, USA).
ChIP-qPCR analysis was also conducted as previously described [28 (link)]. The enriched-DNA was analyzed by quantitative PCR analysis using the Thunderbird SYBR qPCR Mix without ROX (Toyobo, Japan). All EMSA and ChIP-qPCR assays were repeated three times.
For qRT-PCR analysis, Total RNA was extracted with Trizol reagent (Life Technologies, Carlsbad, CA, USA). The genes act and tef were used as references [22 (link)]. The relative normalized transcript level of a gene was computed using the 2−∆∆Ct method [29 (link)]. cDNA synthesis was conducted using the ReverTra Ace qPCR RT Master Mix with a gDNA remover (Toyobo, Japan). qPCR was performed with the Thunderbird SYBR qPCR Mix (no ROX) (Toyobo, Japan). These experiments were repeated three times with three replicates per repeat.
ChIP-qPCR analysis was also conducted as previously described [28 (link)]. The enriched-DNA was analyzed by quantitative PCR analysis using the Thunderbird SYBR qPCR Mix without ROX (Toyobo, Japan). All EMSA and ChIP-qPCR assays were repeated three times.
For qRT-PCR analysis, Total RNA was extracted with Trizol reagent (Life Technologies, Carlsbad, CA, USA). The genes act and tef were used as references [22 (link)]. The relative normalized transcript level of a gene was computed using the 2−∆∆Ct method [29 (link)]. cDNA synthesis was conducted using the ReverTra Ace qPCR RT Master Mix with a gDNA remover (Toyobo, Japan). qPCR was performed with the Thunderbird SYBR qPCR Mix (no ROX) (Toyobo, Japan). These experiments were repeated three times with three replicates per repeat.
7
RNA Isolation, Reverse Transcription, and RT-qPCR Analysis
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Total RNA was isolated from the tissues or cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. Reverse transcription was performed with ReverTra Ace® qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's protocol (20 µl reaction: 14 µl RNA+ 4 µl 5xRT Buffer+ 1 µl Primer Mix+ 1 µl EnzymeMix, step 1 16°C for 5 min, step 2 42°C for 30 min, step 3 98°C for 5 min). The RT-qPCR analysis was performed using the Applied Biosystems QuantStudio 5 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with THUNDERBIRD SYBR qPCR mix (Toyobo Co., Ltd.) according to the manufacturer's protocol (20 µl reaction: 1 µl cDNA+ 7 µl RNA-free water+ 0.8 µl forward primer+ 0.8 µl reverse primer+ 0.4 µl ROX+ 10 µl THUNDERBIRD SYBR qPCR mix, the concentration of primer was 0.2 µM, step 1 95°C for 2 min, step 2 95°C for 15 sec, step 3 60°C for 60 sec, repeat step 2 and step 3 for 40 cycles, final step 72°C for 5 min). GAPDH was used as internal control. The relative expression levels were calculated using the 2−ΔΔCq equation (20 (link)). The primer sequences for PCR were as follows: CITED1, forward 5′-AGGATGCCAACCAAGAGATG-3′ and reverse 5′-GTTTAGTGGGAGGGGTGGTT-3′; GAPDH, forward 5′-GGTCGGAGTCAACGGATTTG-3′ and reverse 5′-ATGAGCCCCAGCCTTCTCCAT-3′.
8
Quantitative RT-PCR of E. coli Transcripts
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RT-qPCR was performed according to standard procedure [48 (link)]. E. coli cells were inoculated into an LB medium at 37 °C under aeration, with constant shaking at 150 rpm. After inoculation for 24 h, the total RNA was extracted using an ISOGEN solution (Nippon Gene). Total RNAs was transcribed to cDNA with random primers using the THUNDERBIRD SYBR qPCR RT Set (TOYOBO, Osaka, Japan). Quantitative PCR (qPCR) was conducted using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) and a LightCycler 96 system (Roche, Barsel, Switzerland). The primer pairs used are described in Table S1c . The cDNA templates were serially diluted 4-fold and used in the qPCR assays. The qPCR mixtures, each containing 10 μL of THUNDERBIRD SYBR qPCR Mix (TOYOBO), 1 μL of each primer (5 μM stock), 7 μL of water, and 1 μL of cDNA, were amplified under the following thermal cycle conditions: 95 °C treatment for 2 min, at 45 cycles of 10 s at 95 °C, and 20 s at 55 °C, and then incubation for 20 s at 72 °C. The 16S rRNA expression level was used to normalize the varying levels of the test samples, and the relative expression levels were quantified using the Relative Quantification software provided by Roche. The results are presented as the average of three independent experiments.
9
RT-qPCR Gene Expression Analysis
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Top individual genes contributing most strongly to the gene-set enrichments were validated using RT-qPCR using M-MLV cDNA synthesis kit (Enzynomics, EZ006). qPCR was performed using THUNDERBIRDTM SYBR® qPCR mix (TOYOBO, QPS-201), CFX96TM Real-Time system (BIO-RAD). Genes and primer-set information are shown below:
10
Quantitative mRNA Expression Analysis
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Total mRNA was extracted from the cells using a Dynabeads mRNA DirectTM Kit (Ambion, Austin, TX), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA remover kit (Toyobo, Osaka, Japan) in accordance with the respective manufacturer’s instructions. Subsequently, specific gene expression was quantified with THUNDERBIRDTM SYBRⓇ qPCR Mix (Toyobo) using a 7500 Real-time PCR system (Applied Biosystems, Foster City, CA). PCR specificity was determined by analyzing the melting curve data. The mRNA levels are presented as 2−ΔCt where Ct=threshold cycle for target amplification and ΔCt=Cttarget gene (specific genes for each sample) – Ctinternal reference (β-actin for each sample). Primer sequences were designed with Primer3 software (Whitehead Institute/MIT Center for Genome Re-search) with mouse cDNA sequence data obtained from GenBank. General information and sequences of primers are described in Supplementary Table S1 .
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