Identification of housekeeping genes useful for the normalization of rna in studies of gene expression in thyroid carcinomas
P Piampiani, C Romei, B Cosci, A Vivaldi, R Ciampi, A Pinchera & R Elisei
Housekeeping genes (HK) are commonly used as controls for the normalization of the RNA amount in quantitative RT-PCR experiments. The question whether their expression is influenced by the metabolic state of the patient and/or the tumour transformation is still unresolved and until now, HK genes have been used in quantitative studies without considering that their expression could be different in tumor with respect to normal tissue. Aim of this study was to identify HK genes equally expressed in the tumor and in the normal tissue of the same patient.
We evaluated the mRNA expression of 5 HK genes (GAPDH, HPRT, β-2-microglobulin, β-actin and cyclophilin A) in 30 papillary thyroid carcinoma (PTC) tissues and in the relative normal thyroid, by Real-time RT-PCR.
RNA was exctracted from the tissues and reverse transcribed into cDNA. After validating the good quality of the cDNA using primers for a specific thyroid gene (PAX-8), samples were used for Real-time RT-PCR using specific primers. The statistical analysis was done by coupling the value of the threshold cycle (Ct) relative to the expression of any single gene in the tumor and in the normal controlateral tissue (t-test paired).
Our results showed that GAPDH and HPRT are equally expressed in the tumor and in the controlateral normal tissue. In particular the Ct values were similar in tumor and benign tissues of 26/30 cases (P=0.25 and P=0.16 respectively). On the contrary, β-2-microglobulin, β-actin and cyclophilin A were significantly more expressed in the normal tissue than in the tumor (P=0.045, P=0.0008 and P=0.033 respectively).
In conclusion, our data suggest that GAPDH and HPRT can both be used for RNA normalization of quantitative studies of gene expression in PTC. At variance, β-2-microglobulin, β-actin and cyclophilin A can not be used since their expression seems to be modulated by tumoral transformation.