Total RNA was isolated from the cells using the Easy-Spin™ Kit (iNtRON) in accordance with the manufacturer’s protocol. The cDNA was synthesized by using a ReverTra Ace qPCR RT Mix (TOYOBO). The qRT-PCR was performed by using iTaq™ Universal SYBR™ Green Supermix (Bio-Rad). Used Primers are listed in Table 1 . During PCR, a dissociation curve was constructed in the range of 65°C to 95°C, and the cycling parameters of qPCR were followed; 1 cycle for 1 min at 95°C, 40 cycles for 15 s at 95°C, and 1 min at 60°C. The GAPDH was used as an internal control to normalize the variability in target gene expression. Statistical analyses on three readings were carried out using Student’s t-test, and p values of less than 0.05 were considered significant.
Revertra ace qpcr rt mix
Manufactured by Toyobo
Sourced in Japan
ReverTra Ace qPCR RT Mix is a real-time reverse transcription polymerase chain reaction (RT-qPCR) reagent. It is designed for the reverse transcription and subsequent quantitative PCR amplification of RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Primescript 4 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Total rna isolation mini kit, by Agilent Technologies (1 mentions)
Gdna remover, by Toyobo (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Thunderbird sybr qpcr mix, by Toyobo (1 mentions)
Mx3005p real time pcr system, by Agilent Technologies (1 mentions)
Pmd19 vector, by Takara Bio (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
5 protocols using revertra ace qpcr rt mix
1
Quantitative RT-PCR Analysis of Gene Expression
2
Quantitative RT-PCR Analysis of Gene Expression
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Total RNAs from MS1 cells or EMRECs were isolated using Sepasol I‐RNA I Super G (Nacalai Tesque), the RNeasy Plus Mini kit (QIAGEN, Hilden, Germany), or the RNeasy FPE Kit (QIAGEN) and reversely transcribed using the PrimeScript II 1st strand cDNA Synthesis Kit (TaKaRa Bio, Otsu, Japan), the PrimeScript IV 1st strand cDNA Synthesis Kit (TaKaRa Bio), or ReverTraAce qPCR RT Mix (TOYOBO, Osaka, Japan). The quantitative RT‐PCR (qRT‐PCR) was performed with the Step One Plus Real‐Time PCR System (Applied Biosystems, Waltham, MA, USA) or QuantStudio 3 (Applied Biosystems) using gene‐specific primers and Fast Start Universal SYBR Green Master (Roche, Basel, Switzerland) or PowerUp SYBR Green Master Mix (Applied Biosystems). All expression data were normalized to the expression of β‐actin. The primers used for qRT‐PCR are listed in Table S1 .
3
Quantitative PCR for Gene Expression Analysis
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Each culture of cells was grown for 4 h and total RNA was extracted using a Total RNA Isolation Mini Kit (Agilent Technologies). The concentration of total RNA was determined using a Nano Vue Plus spectrophotometer (Life Technologies, Carlsbad, CA). The quality was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA was generated from the extracted total RNA by reverse transcription using ReverTra Ace qPCR RT Mix and gDNA Remover (Toyobo, Osaka, Japan). Quantitative PCR experiments were carried out in triplicate using Thunderbird SYBR qPCR Mix (Toyobo) and an Mx3005P Real Time PCR system (Agilent Technologies). Specific primers for the amplification of the selected genes and ACT1 as a house-keeping internal-standard gene are listed in Supplementary Table 1 . The fold-change of the transcript levels was calculated using the 2−∆∆CT method24 (link).
4
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from cells or spheroids using Easy-Spin Kit (Intron), and cDNA was synthesized using ReverTra Ace qPCR RT Mix (Toyobo). The qRT-PCR was conducted with iTaq Universal SYBR Green Supermix (Bio-Rad) using specific primers (Table 1 ). qPCR amplifying condition consisted of 1 cycle for 30 sec at 95°C, and 40 cycles for 15 sec at 95°C of denaturation and 60 sec at 55-60°C of annealing/extension. A melt curve was constructed in the range of 65°C to 95°C with 0.5°C increments per step. The relative comparison of each gene was analyzed, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalizing gene expression as an internal control. Three samples were analyzed for each target gene.
5
Cloning and Characterization of Cowpea Genes
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A cowpea cDNA template was prepared from mRNA isolated from roots using the RNeasy Plant Mini Kit (Qiagen) and ReverTra Ace qPCR RT Mix with genomic DNA (gDNA) Remover (TOYOBO) for RT-PCR. The full-length VuMAX1, VuCYP722C, and VuCYP728B cDNAs were amplified using the PrimeSTAR HS DNA polymerase (TaKaRa Bio) with primer sets shown in table S3. The primers were designed using the complete CDS sequences recorded in the Phytozome database (Vigna unguiculata v1.0; NSF, UCR, USAID, DOE-JGI; http://phytozome.jgi.doe.gov/ ). A synthetic SlCYP722C (Solyc02g084930.3) cDNA was obtained from Eurofins Genomics. Each amplicon was cloned into the pMD19 vector (Takara Bio) according to the manufacturer’s instructions. Table S4 shows gene sequences characterized in this study.
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