MiRNAs were extracted from cells by using the method described in our previous study 5 (link). Taqman probes (miR-93, U6) were acquired from the Taqman miRNA assay (Life Technologies, Carlsbad, CA, USA). Subsequent detection by qPCR was performed using the TaqMan PCR Master Mix (TAKARA, Dalian, China) with the StepOne Plus Real Time PCR Detection System (Life Technologies). MiR-93 expression was quantified by the relative quantification method with U6 as the reference gene and was verified by comparing with the control cells.
Taqman pcr master mix
Manufactured by Takara Bio
Sourced in China
The TaqMan PCR Master Mix is a ready-to-use solution for real-time PCR amplification. It contains all the necessary components for efficient DNA/RNA detection and quantification, including a thermostable DNA polymerase, dNTPs, and optimized buffer system.
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2 protocols using taqman pcr master mix
1
Quantifying miRNA Expression by qPCR
2
Real-Time PCR Quantification of Viral RNA
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Infected DCs were harvested from the different treatments. Total RNA was extracted from the DCs using RNAios Plus (Takara, Dalian, China). Reverse transcription of the RNA was performed with the designed primers (Table 1 ), which amplified fragments of the target genes. In this experiment, 2 μl of template RNA was subjected to a real-time PCR reaction with a final volume of 20 μl using the Taq-Man PCR Master Mix (Takara, Dalian, China). The thermal cycling conditions were 5 min at 95°C, followed by 40 cycles of 15 s at 95°C and 34 s at 60°C using the Applied Biosystems 7500 real-time PCR system. The −ΔΔCT method was used for the relative virus quantification.
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