RNA was isolated by RNA Purification Kit (Cat#RN001, EZBioscience) and reverse transcribed by HiScript III RT SuperMix reverse-transcription reagent kit (Cat#711-02/03, Vazyme Biotech Co.). qPCR was performed on a LightCycler 96 Real-Time PCR System (F. Hoffmann-La Roche) using FastStart Essential DNAGreen Master assay (F. Hoffmann-LaRoche). The primers used are listed in Supplementary Table 1 . All of the primers were synthesized by Sangon, Shanghai.
Faststart essential dnagreen master assay
Manufactured by Roche
The FastStart Essential DNAGreen Master assay is a real-time PCR (polymerase chain reaction) reagent kit designed for amplification and quantification of DNA. It contains all the necessary components, including a DNA polymerase enzyme, buffer, and a fluorescent dye, to perform real-time PCR reactions. The assay is suitable for various applications that require sensitive and accurate DNA quantification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Lightcycler 96 real time pcr system, by Roche (3 mentions)
Hiscript 3 rt supermix reverse transcription reagent kit, by Vazyme (2 mentions)
Rna purification kit, by EZBioscience (1 mentions)
Primescript reverse transcription reagent kit, by Takara Bio (1 mentions)
3 protocols using faststart essential dnagreen master assay
1
RNA Quantification and Analysis
2
Chemerin gene expression analysis
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Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific). 1 μg of RNA and PrimeScript reverse transcription reagent kit (TaKaRaBio, Shiga, Japan) were used to perform reverse transcription. Real-time qPCR was performed on a LightCycler 96 Real-Time PCR System (F. Hoffmann-La Roche) using FastStart Essential DNAGreen Master assay (F. Hoffmann-La Roche). The 2ΔΔCt method was used for semiquantitative analysis. GAPDH served as an internal standard. Primers were used as follows: chemerin-forward, 5′-GCAGACAAGCTGCCGGA-3′; chemerin-reverse, 5′-AGTTTGATGCAGGCCAGGC-3′; GAPDH-forward, 5′-ACG GATTTGGTCGTATTGGG-3′; GAPDH-reverse, 5′-TGATTTT GGAGGGATCTCGC-3′.
3
Adipose Tissue RNA Extraction and RT-qPCR
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Total RNA from adipose tissue was extracted with Trizol reagent (Thermo Fisher Scientific). Reverse transcription was performed using 1 ug of RNA and HiScript III RT SuperMix reverse-transcription reagent kit (Vazyme Biotech Co., Ltd, Nanjing, China). Real-time qPCR was performed on a LightCycler 96 Real-Time PCR System (F. Hoffmann-La Roche) using FastStart Essential DNAGreen Master assay (F. Hoffmann-LaRoche). The 2−ΔΔCt method was used for semiquantitative analysis. 18s rRNA and 36B4 served as internal standard. Primers are listed in Supplemental Table 2 .
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