The pseudogene problem and RT-qPCR data normalization; SYMPK: a suitable reference gene for papillary thyroid carcinoma.

In RT-qPCR, accuracy requires multiple levels of standardization, but results could be obfuscated by human errors and technical limitations. Data normalization against suitable reference genes is critical, yet their observed expression can be confounded by pseudogenes. Eight reference genes were selected based on literature review and analysis of papillary thyroid carcinoma (PTC) microarray data. RNA extraction and cDNA synthesis were followed by RT-qPCR amplification in triplicate with exon-junction or intron-spanning primers. Several statistical analyses were applied using Microsoft Excel, NormFinder, and BestKeeper. In normal tissues, the least correlation of variation (CqCV%) and the lowest maximum fold change (MFC) were respectively recorded for PYCR1 and SYMPK. In PTC tissues, SYMPK had the lowest CqCV% (5.16%) and MFC (1.17). According to NormFinder, the best reference combination was SYMPK and ACTB (stability value = 0.209). BestKeeper suggested SYMPK as the best reference in both normal (r = 0.969) and PTC tissues (r = 0.958). SYMPK is suggested as the best reference gene for overcoming the pseudogene problem in RT-qPCR data normalization, with a stability value of 0.319.