To quantify the
transcript level of the TP53 gene, quantitative (q) PCR was performed.
To perform this process, Rotor-Gene Q real-time PCR detection system
(QIAGEN) was used. GAPDH was used as an endogenous control for normalization.
Dilutions were made for each concentrated cDNA sample. For target
and housekeeping gene, real-time PCR was performed using 2X Maxima
SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific). PCR conditions
were optimized as 95 °C for 3 min, followed by 35 cycles at 95
°C for 30 s, 59 °C for 15 s, and 72 °C for 15 s and
further placed at 72 °C for 10 min. Quantitative PCR analysis
was also carried out for positive and negative controls in each run
to check the machine and all the reaction conditions.
GAPDH
was also amplified for normalization and taken as an endogenous control.
The Ct value was analyzed for each sample using the Livak method.15 (link) The Livak method describes the fold change in
gene expression after performing real-time PCR.
transcript level of the TP53 gene, quantitative (q) PCR was performed.
To perform this process, Rotor-Gene Q real-time PCR detection system
(QIAGEN) was used. GAPDH was used as an endogenous control for normalization.
Dilutions were made for each concentrated cDNA sample. For target
and housekeeping gene, real-time PCR was performed using 2X Maxima
SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific). PCR conditions
were optimized as 95 °C for 3 min, followed by 35 cycles at 95
°C for 30 s, 59 °C for 15 s, and 72 °C for 15 s and
further placed at 72 °C for 10 min. Quantitative PCR analysis
was also carried out for positive and negative controls in each run
to check the machine and all the reaction conditions.
GAPDH
was also amplified for normalization and taken as an endogenous control.
The Ct value was analyzed for each sample using the Livak method.15 (link) The Livak method describes the fold change in
gene expression after performing real-time PCR.
