Severe hypercalcemia caused by parathyroid hormone in a rectal cancer metastasis: a case report

Total RNA was isolated from FFPE tissue sections using the High Pure FFPE tissue RNA Isolation kit (Roche) according to the manufacturer’s recommendation. RNA concentration was measured by Qubit (Thermo Fisher Scientific). Complementary deoxyribonucleic acid (cDNA) synthesis of total RNA was performed with SuperScript™ IV Reverse Transcriptase (ThermoFisher Scientific). 2.5 μM of random hexamer primer (ThermoFisher Scientific) and approximately 80 ng of template were used for the reaction. Total RNA was denaturated at 65 °C for 5 min, and cDNA was synthesized at 53 °C for 10 min. For real-time quantitative polymerase chain reaction (RT-qPCR), cDNA was mixed with FastStart Essential DNA Green Master (Roche Life Science) and 0.25 μM forward and reverse primer. All primer sequences are provided in Supplementary Table 1. The LightCycler® 96 was used for quantification, and the ΔΔCq-method was used to calculate fold change using the geometric mean of housekeeping genes (GAPDH and RPLPO) as internal reference.