Maxima fluorescein rt qpcr master mix

Total RNA was isolated from human thyroid specimens and thyroid cancer cell lines using an RNA Mini Kit (A&A Biotechnology, Poland) according to the recommended protocol, and the RNA integrity was verified by agarose gel electrophoresis. Total RNA (1 µg) was used for cDNA synthesis with a High Capacity cDNA Reverse Transcription Kit (Life Technologies, Applied Biosystems, USA). Expression of the human PDPN and 18S rRNA genes was quantified by RT-qPCR using the cDNAs as template in reactions containing the double-stranded DNA-specific dye SYBR Green I and Maxima Fluorescein RT-qPCR Master Mix (Thermo Scientific, Canada), and specific oligonucleotide primers (listed below), as described previously [24] (link).
PDPN (NM_006474)
Forward: 5′-CGAAGATGATGTGGTGACTC-3′Reverse: 5′-CGATGCGAATGCCTGTTAC-3′18S rRNA (NM_02255)
Forward: 5′-CCAGTAAGTGCGGGGTCATAAG-3′Reverse: 5′-CCATCCAATCGGTAGTAGCG-3′.
Amplification, data acquisition and data analysis were performed using the iQ5 Real-Time PCR Detection System and software (Bio-Rad, USA).

Expression of the human genes was quantified by RT-qPCR, where the cDNAs served as a template using Maxima Fluorescein RT-qPCR Master Mix (ThermoFisher Scientific Waltham, MA, USA), containing the double-stranded DNA-specific dye SYBR Green I, and specific oligonucleotide primers (primer sequences for RT-qPCR are presented in Supplementary Table S1). PCR reactions were performed in triplicates with the following conditions: 95 °C/30 s, 40 cycles of 95 °C/5 s, 58 °C/15 s and 72 °C/10 s in iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The Ct values estimated for analyzed genes were normalized against corresponding Ct values of β-ACTIN.