Discovery ultra

mRNA ISH, a novel method to detect mRNA in FFPE tissues,^{26 (link)} was performed for TERT mRNA on a Discovery Ultra automation system (Ventana Medical Systems, Inc.) by using RNAscope® VS Reagent Kit – RED (Advanced Cell Diagnostics). VS Probe – Hs-TERT (Cat#605516) specific to the sequence region between nucleotides 2164 to 3231 encoding the TERT transcript was used according to the manufacturer’s instructions. Briefly, 4-µm FFPE tissue sections of tumors were pretreated in citrate buffer with heat, followed by protease digestion before hybridization with the target oligo probes. Slides were hybridized sequentially with target probes incubated at 43°C for 2 h and 32 min, preamplifier at 53°C for 32 min, and amplifier at 53°C for 32 min, and label probes at room temperature for 12 min. Between the hybridization steps, slides were washed with Ribowash buffer (0.1× saline sodium citrate). Hybridization signals were detected by chromogenic development with Fast Red, followed by counterstaining with hematoxylin. Each sample was quality controlled for RNA integrity with an RNAscope probe for PPIB RNA and for background with a probe for bacterial dapB RNA. The specific RNA staining signal was identified as intracellular red punctate dots.