Analysis of mRNA and miRNA levels was performed on the StepOnePlus Real-Time PCR System (Applied Biosystems). For mRNA detection, cDNA was generated with the QuantiTect Reverse Transcription Kit (Qiagen). Diluted cDNA samples were amplified to establish a standard curve for calculation of relative target concentrations using Express SYBR GreenER qPCR SuperMix with Premixed ROX (Life Technologies). The housekeeping gene ACTB was used as an internal control. The primers for the genes of interest were synthesized by RealTimePrimers.com or Sigma-Aldrich (Extended Data Table 9 ). Analysis of lncRNA and miRNA levels was performed with the use of the TaqMan fast advanced master mix (Applied Biosystems). TaqMan lncRNA, miRNA, and Pri-miRNA expression assays (Life technologies) were used according to the manufacturer's instructions, with ACTB or U6 small nuclear RNA (U6 snRNA) as the internal control (Extended Data Table 10 ). The relative expression of RNAs was calculated using the comparative Ct method.
Express sybr greener qpcr supermix with premixed rox
Manufactured by Thermo Fisher Scientific
The Express SYBR GreenER qPCR SuperMix with Premixed ROX is a ready-to-use reaction mix for quantitative real-time PCR (qPCR) applications. It contains all the necessary components, including SYBR Green I dye, a passive reference dye (ROX), and a chemically-modified hot-start DNA polymerase.
Automatically generated - may contain errors
Lab products found in correlation
Pri mirna expression assays, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
3 protocols using express sybr greener qpcr supermix with premixed rox
1
Quantitative Analysis of RNA Levels
2
Quantitative Analysis of RNA Levels
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Analysis of mRNA and miRNA levels was performed on the StepOnePlus Real-Time PCR System (Applied Biosystems). For mRNA detection, cDNA was generated with the QuantiTect Reverse Transcription Kit (Qiagen). Diluted cDNA samples were amplified to establish a standard curve for calculation of relative target concentrations using Express SYBR GreenER qPCR SuperMix with Premixed ROX (Life Technologies). The housekeeping gene ACTB was used as an internal control. The primers for the genes of interest were synthesized by RealTimePrimers.com or Sigma-Aldrich (Extended Data Table 9 ). Analysis of lncRNA and miRNA levels was performed with the use of the TaqMan fast advanced master mix (Applied Biosystems). TaqMan lncRNA, miRNA, and Pri-miRNA expression assays (Life technologies) were used according to the manufacturer's instructions, with ACTB or U6 small nuclear RNA (U6 snRNA) as the internal control (Extended Data Table 10 ). The relative expression of RNAs was calculated using the comparative Ct method.
3
Quantitative RNA Expression Analysis
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Total RNA was extracted using a RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using a PrimeScript RT reagent kit (TaKaRa). SYBR Premix Ex Taq II (TaKaRa) was used to amplify the double-stranded cDNA of interest. RT-qPCR primers for the genes of interest were synthesized by TaKaRa. The levels of GAPDH were used as internal controls. A standard curve was established by amplifying diluted cDNA samples for calculation of relative target concentrations using Express SYBR GreenER qPCR SuperMix with Premixed ROX (Life Technologies). The 2–ΔΔCt method was used to determine the relative expression level of RNA between groups. The primer sequences used in this study are listed in Supplementary Table 5 .
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