Cells were harvested and extracted mRNA using the miRNAeasy Mini Kit (QIAGEN, Germantown, MD, USA, 217004) following the company’s instructions. After extracting mRNA, reverse-transcription (cDNA synthesis kit (Quantabio 101414-106), and real time PCR (SYBR green RT-PCR reagents kit (Quantabio, Beverly, MA, USA, 101414-276) were used. The condition for the real-time PCR machine (Quantabio) is as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The fold changes were calculated based on 18S in the threshold cycle (Cq). The sequence primers are shown in Table 1 .
Real time pcr machine
Manufactured by Quantabio
The Real-time PCR machine is a laboratory instrument designed for the amplification and detection of DNA or RNA sequences in real-time. It accurately measures the accumulation of amplified product during the PCR reaction, providing quantitative data on the initial target concentration.
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2 protocols using real time pcr machine
1
mRNA Extraction and Real-Time PCR Analysis
2
Quantitative Real-Time PCR Protocol
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The extraction of RNA was performed with the miRNAeasy Mini Kit (Qiagen). Reverse transcription of RNA was carried out using the “cDNA Synthesis kit” (OriGene). The PCR was performed with a master mix, containing SYBR green (Applied Biosystems). The real‐time PCR machine (Quantabio) was programmed as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Upon completion of the run, data analysis was performed (delta–delta Ct method). Genes of interest were normalized to 18S. Fold changes relative to the controls are provided. Forward and reverse primers are given in Table 1 .
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