Total RNA was extracted from the liver tissues using TRIpure reagent (Aidlab Biotechnologies Co., Ltd., Beijing, China) and reverse transcribed into cDNA was synthesized using a FastQuant RT kit (Tiangen Biotech Co. Ltd., Beijing, China). The mRNA expression levels were measured on a CFX96 real-time PCR detection system (CFX96, Bio-Rad, United States) using SuperReal PreMix Plus with SYBR Green (Tiangen Biotech Co. Ltd., Beijing, China). The primer sequences were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and are provided in Table 2 . The mRNA expression levels were normalized to GAPDH and calculated by the 2–ΔΔCt method.
Superreal premix plus with sybr green
Manufactured by Tiangen Biotech
Sourced in China
SuperReal PreMix Plus with SYBR Green is a ready-to-use solution for real-time PCR amplification and detection. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, enabling real-time monitoring of DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Tripure reagent, by Aidlab (2 mentions)
Fastquant rt kit, by Tiangen Biotech (2 mentions)
Cfx96 qrt pcr detection system, by Bio-Rad (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Real time pcr detection system cfx 96, by Bio-Rad (1 mentions)
Primer premier 6, by Premier Biosoft (1 mentions)
Total rna, by Takara Bio (1 mentions)
Beacon designer 8, by Premier Biosoft (1 mentions)
Qrt pcr, by Sangon (1 mentions)
5 protocols using superreal premix plus with sybr green
1
Quantifying Hepatic Gene Expression
2
Quantitative Real-Time PCR Protocol
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qRT-PCR was implemented using the Kit (SuperRealPreMix Plus with SYBR Green from TIANGEN, Beijing, China) on a Real-Time PCR Detection System CFX96 (Bio-Rad, Hercules, CA USA). In addition, all cDNA templates used in the experiment were of the same concentration. Twenty microliters reaction systems were preparing using with following: 1 μL of the cDNA template, 7.4 μL of water, 10 μL of 2× SuperRealPreMix Plus, 0.4 μL of 50× ROX Reference Dye, and 0.6 μL of the forward and reverse primers. The PCR program involved a two-step process that was run for 40 cycles: 95 °C for 10 min, then denaturation at 95 °C for 10 s, annealing at 60 °C for 32 s, and extension at 72 °C for 10 s. Each reaction had four replicates. Melting curve data were gathered from 65 °C to 95 °C in 0.5 °C increments. The standard curve of each primer pair was established with serial dilutions of cDNA ((1/5)0, (1/5)1, (1/5)2, (1/5)3, (1/5)4 and (1/5)5). The amplification efficiency (E) of qRT-PCR was determined according to the equation: E = 10−1/K, where K represents the slope of the standard curve.
3
Validating Gene Expression by RT-qPCR and RNA-Seq
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Gene expression profiles were determined by qRT-PCR on a CFX96 qRT-PCR detection system (Bio-Rad, Hercules, CA, USA) with associated software. cDNA reverse-transcribed from total RNA (Takara, Shiga, Japan) was used as the template along with SuperReal PreMix Plus with SYBR Green (Tiangen Biotech, Beijing, China) and gene-specific primers designed with Primer Premier 6 software (Premier Biosoft, Palo Alto, CA, USA). Relative abundance of the transcripts was calculated by the comparative cycle threshold method with Beta-actin used as an internal reference gene [13 ]. Reactions were prepared in triplicate for each sample. Relative expression levels were calculated by the 2−ΔΔCT method. The expression value of each gene from qRT-PCR and RNA-seq was log2 transformed and performed by linear regression analysis. A linear regression coefficient (r) was obtained between the results of RT-qPCR and RNA-Seq.
4
Quantification of M. purpureus Gene Expression
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M. purpureus M1-36 and M1 gene expression profiles were determined by qRT-PCR on a CFX96 qRT-PCR detection system (Bio-Rad, Hercules, CA, USA) with associated software.
cDNA reverse-transcribed from total RNA was used as the template along with SuperReal PreMix Plus with SYBR Green (Tiangen Biotech, Beijing, China) and unigene-specific primers (Table 1 ) designed with Beacon Designer 8 software (Premier Biosoft). The amplification program was as follows: 95°C for 15 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Relative abundance of the transcripts was calculated by the comparative cycle threshold method with GAPDH used as an endogenous control. Reactions were prepared in triplicate for each sample.
cDNA reverse-transcribed from total RNA was used as the template along with SuperReal PreMix Plus with SYBR Green (Tiangen Biotech, Beijing, China) and unigene-specific primers (
5
Puerarin and Fenofibrate Biochemical Assays
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Puerarin (purity 98%) was purchased from Nanjing Jingzhu Bio-technology Co., Ltd. (Nanjing, China). Fenofibrate was bought from Recipharm Fontaine (France). The commercial kits of total cholesterol (TC), TG, aspartate transferase (AST), alkaline phosphatase (ALP), and alanine transaminase (ALT) were obtained from Biosino Bio-Technology and Science Inc. (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits of FFA, VLDL, acetyl-CoA, acetoacetate (AcAc), and succinate dehydrogenase (SDH) were purchased from Beijing Sino-UK Institute of Biological Technology (Beijing, China). The assay kits of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA), and enzyme-linked immunosorbent assay (ELISA) kits of TNF-α, interleukin-18 (IL-18) and interleukin-1β (IL-1β) were bought from Beijing Sino-UK Institute of Biological Technology (Beijing, China). TRIpure reagent was obtained from Aidlab Biotechnologies Co., Ltd. (Beijing, China). FastQuant RT Kit and SuperReal PreMix Plus with SYBR Green were purchased from Tiangen Biotech Co. Ltd. (Beijing, China). The primers of quantitative real-time PCR (qRT-PCR) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
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