Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied to verify the expression of the target LIMD2 in 20 pairs of ECA and adjacent normal esophageal tissues. The primers of LIMD2 were purchased from BioSune (Shanghai, China) (Table 2 ). An RT2162; All-in-One Mix with dsDNase (Monad Biotech Co. Ltd., Shanghai, China) was used to synthesize cDNA from 1 µg of total RNA. The qRT-PCR analyses were conducted on the Quant Studio 6 Flex qRT-PCR system (Applied Biosystems, Thermo Fisher Scientific Co. Ltd., United States) using the Hieff ®qPCR SYBR® Green Master Mix, Low Rox (Yeasen, Biotechnology Co., Ltd, Shanghai, China). The reaction was: 95°C for 10 min, then 40 cycles of 95°C for 15 s and 6°C for 1 min. The reference gene was GAPDH, and the relative gene expression levels were calculated using the 2−ΔΔCt method.
Hieff qpcr sybr green master mix low rox
Manufactured by Yeasen
Sourced in China
Hieff® qPCR SYBR® Green Master Mix (Low Rox) is a ready-to-use solution designed for quantitative real-time PCR (qPCR) experiments. It contains SYBR® Green I dye, DNA polymerase, dNTPs, and low-concentration ROX reference dye.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 6 flex qrt pcr system, by Thermo Fisher Scientific (1 mentions)
Abi 7500 pcr detection system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Beyotime (1 mentions)
Abi prism 7300 system, by Thermo Fisher Scientific (1 mentions)
Hifair 3 1st strand cdna synthesis supermix for qpcr gdna digester plus, by Yeasen (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by PerkinElmer (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Maclin Biochemical Technology (1 mentions)
Hifair 2 1st strand cdna synthesis supermix for qpcr, by Yeasen (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Hepg2 cells, by Sangon (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Maclin Biochemical Technology (1 mentions)
Trizol, by Takara Bio (1 mentions)
Hifair 2 1st strand cdna synthesis kit, by Yeasen (1 mentions)
5 protocols using hieff qpcr sybr green master mix low rox
1
Quantitative Analysis of LIMD2 Expression in Esophageal Cancer
2
RT-qPCR Assay for Transcript Quantification in Tuta absoluta
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Relative standard curves for the transcripts were generated with 2-fold serial dilutions of cDNA (1/2, 1/4, 1/8, 1/16, 1/32, and 1/64) extracted from the second instar larvae of T. absoluta. The corresponding RT-qPCR efficiencies (E) were determined for each gene and calculated according to the following equation: E = (10[−1/slope] − 1) × 100 [49 (link)]. RT-qPCR was performed on the ABI 7500 PCR Detection System (Applied Biosystems, Waltham, MA, USA). The RT-qPCR reactions consisted of 10 µL Hieff® qPCR SYBR® Green Master Mix (Low Rox) (Yeasen Biotech Co., Ltd., Shanghai, China), 0.4 µL each forward and reverse primers (10 µM), and 1 µL the cDNA template. Thermal cycling conditions were as follows: an initial cycle at 95 °C (30 s), followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. After all reactions, a melting curve analysis from 65 to 95 °C was used to ensure the consistency and specificity of the amplified product. According to the results of the standard curve, the cDNA template of all samples was diluted 5 times in the reference gene expression experiment. Each treatment included five biological replicates, and each reaction was performed in triplicate.
3
Quantitative Analysis of Prostate Cancer RNA
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We first used a TRIzol reagent (Beyotime, Jiangsu, China) to extract total RNA from various prostate cancer cells. These total RNA were then reversely transcribed into cDNA using the Hifair®III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (YEASEN, Shanghai, China), which was subsequently detected by qPCR using the Hieff® qPCR SYBR® Green Master Mix (Low Rox) (YEASEN, Shanghai, China) an ABI Prism 7300 system (Thermo Fisher Scientific). In this experiment, GAPDH was used as an internal reference gene, and all primer sequences are shown in Table S1 .
4
Cytotoxicity Evaluation of OA and PIP
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HepG2 cells were obtained from Sangon Biotech Co., Ltd. (D611027-0001, Shanghai, China) and cultured in Dulbecco’s modified Eagle medium (DMEM) medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 KU/L penicillin (Gibco, Carlsbad, CA, USA) and 100 μg/mL streptomycin (Gibco, Carlsbad, CA, USA). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (purity~98%) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) (purity~99%) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) and Hieff® qPCR SYBR Green Master Mix (Low Rox) were purchased from Yeasen Biotechnology Co., Ltd. (Shanghai, China).
HepG2 cells were incubated overnight in 96-well plates at a density of 1 × 106 cells/well. The cells were cultured with OA and PIP at different concentrations for 24 h. Subsequently, the final concentration of 0.5 mg/mL MTT was added and incubated at 37 °C for 4 h. After removing the MTT from each well, the insoluble formazan crystals were dissolved by DMSO, and the absorbance was measured with a microplate reader (PerkinElmer, Waltham, MA, USA) at 490 nm.
HepG2 cells were incubated overnight in 96-well plates at a density of 1 × 106 cells/well. The cells were cultured with OA and PIP at different concentrations for 24 h. Subsequently, the final concentration of 0.5 mg/mL MTT was added and incubated at 37 °C for 4 h. After removing the MTT from each well, the insoluble formazan crystals were dissolved by DMSO, and the absorbance was measured with a microplate reader (PerkinElmer, Waltham, MA, USA) at 490 nm.
5
Quantifying Gene Expression in Periwinkle
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Two-step qPCR was performed to quantify NPR1-GFP gene, PR genes, and ICS gene expression in periwinkle leaves with the following commercially available kits: Trizol (Takara, Tokyo, Japan) for total RNA extraction from leaves, Hifair® Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai, China) to reverse transcribe total RNA into cDNA, and Hieff® qPCR SYBR Green Master Mix (Low Rox) (Yeasen Biotechnology, Shanghai, China) for qPCR. The 18S rDNA gene was used as a reference gene in all qPCR experiments. The sequences of the primers used were collated in Table S1 . The qPCR conditions were as follows: 94°C for 2 min, followed by 30 cycles of 94°C for 20 s, 60°C for 20 s, and 72°C for 20 s. The qPCR data were analyzed by the ddCt method to obtain the normalized gene expression fold change with respect to the 18S rDNA housekeeping gene and control sample (Li et al., 2019c (link); Demirer et al., 2019 (link)). For each sample, qPCR was performed as three technical replicates (three reactions from the same isolated RNA batch), and the entire experiment consisting of independent infiltrations and RNA extractions from different plants was repeated three times (three biological replicates).
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