TERT Expression Quantification in Breast Tumor FFPE Samples

RNA extraction was performed for those patients’ samples in which tumor tissue from the same FFPE block was available. Total RNA was isolated using the RNeasy FFPE Kit (Qiagen, 73,504), following the manufacturer’s recommendations and then was reverse transcribed using SuperScript IV Reverse Transcriptase, following the manufacturer’s instructions (Invitrogen, 8,090,010). hTERT gene expression estimation was performed using the Droplet Digital PCR (ddPCR) technology, which has been proven to provide more precise and reproducible results in FFPE samples [73 (link)].
The QX200 Droplet Digital PCR (ddPCR) system (Bio-Rad Laboratories, CA, USA) and Taqman probes (Life Technologies, USA), TERT probe Hs00972650_m1 and TBP probe Hs00427621_m1, as an endogenous control, were used in a duplex reaction mode. Different controls (no template, no reverse transcriptase (RT), and Human Universal RNA) were run in parallel with the study samples and the data was analyzed using Quanta-Soft v1.4 (Bio-Rad Laboratories). The TERT/TBP ratio of clinical breast FFPE samples was determined for each sample [74 (link), 75 (link)]. Then, the obtained ratios were calibrated for HeLa cell line transcript ratios. The results obtained represent the expression of breast tumor samples relative to HeLa cell line [22 (link)].