Qscript cdna xlt cdna synthesis supermix

Synthesis of first-strand cDNAs was performed with the above mentioned total RNA (1ug), and random hexamer primers using qScript cDNA XLT cDNA Synthesis SuperMix (Quanta Biosciences, Inc.) following instructions. Real-time PCR was performed using the Fast SYBR Green Master Mix on a StepOnePlus™ Real-time PCR System (Applied Biosystems) with a primer concentration of 300 nM. Primer sequences and the related gene Accession Number are listed in Table 1. Reaction conditions consisted of 95°C for 20 sec. followed by 40 cycles of 95°C for 3 sec. 60 °C for 30 sec. Single PCR product was confirmed with the heat dissociation protocol at the end of the PCR cycles. Human α-tubulin was used as controls to normalize the starting quantity of RNA. Quantitative values were obtained from the threshold PCR cycle number (CT) at which point the increase in signal associated with an exponential growth for PCR product starts to be detected. The target mRNA abundance in each sample was normalized to its endogenous control level and the relative mRNA expression levels were analyzed using the ΔΔCT method. Reaction of each sample was performed in triplicate.

Synthesis of first-strand cDNAs was performed with the above mentioned total RNA (1ug), and random hexamer primers using qScript cDNA XLT cDNA Synthesis SuperMix (Quanta Biosciences, Inc.) following instructions. Real-time PCR was performed using the Fast SYBR Green Master Mix on a StepOnePlus^™ Real-time PCR System (Applied Biosystems) with a primer concentration of 300 nM. Primer sequences and the related gene Accession Number are listed in Table 1. Reaction conditions consisted of 95°C for 20 sec, followed by 40 cycles of 95°C for 3 sec. 60 °C for 30 sec. Single PCR product was confirmed with the heat dissociation protocol at the end of the PCR cycles. Human α-tubulin was used as controls to normalize the starting quantity of RNA. Quantitative values were obtained from the threshold PCR cycle number (CT) at which point the increase in signal associated with an exponential growth for PCR product starts to be detected. The target mRNA abundance in each sample was normalized to its endogenous control level and the relative mRNA expression levels were analyzed using the ΔΔCT method. Reaction of each sample was performed in triplicate.