Experimental validation using qRT-PCR was performed to demonstrate the reliability of the bioinformatics analyses applied. From the top 25 positively DE lncRNAs and from the top 20 negatively DE lncRNAs, it were selected for validation those lncRNAs with low adjusted p-values to minimize expression variability, especially in the comparison BRAFWT × BRAFV600E tumor, among others characteristics (for detailed information see Methods). From a total of 5 DE lncRNAs selected for validation from the TCGA analysis (Fig. 2 and Supplemental Fig. S1, upper part), 4 DE lncRNAs were validated using thyroid cell lines (Fig. 2 and Supplemental Fig. S1, lower part), which strengths the reliability of this bioinformatics analysis, although the experimentally tested set of lncRNAs constitutes a relatively small sampling. Considering all comparisons, we obtained a very expressive validation efficiency [from a total of 8 different comparisons (Normal × Tumor and BRAFWT × BRAFV600E), 6 were validated]. Downregulation of ENSG00000235070.3 and ENSG00000255020.1 in PTC was confirmed in the tumor cell lines TPC1 (BRAFWT) and BCPAP (BRAFV600E) compared to the normal immortalized cell line NTHY (Fig. 2). Also, downregulation of their expression was in accordance to the bioinformatics analysis, since lower expression for both of them was observed in BCPAP (BRAFV600E) compared to TPC1 (BRAF wild type) (Fig. 2). Noteworthy, is that due to the very low abundance of ENSG00000255020.1 in the BCPAP cell line, qRT-PCR resulted in two unspecific melting peaks, which did not influenced the results. Upregulation of ENSG00000273132.1 in PTC was confirmed, however its overexpression in BRAFV600E tumors was not observed in the cell line BCPAP compared to TPC1 (Fig. 2), maybe due to the small log2 fold change value (1.69) of this comparison. Overexpression of ENSG00000230498.1 in BRAFV600E PTC compared to BRAF wild type tumors was also confirmed (Supplemental Fig. S1); nevertheless ENSG00000247311.2 was undetectable in both TPC1 and BCPAP cells (Supplemental Fig. S1).Figure 2Experimental validation of DE lncRNAs. Upper part of panel displays the expression levels of the indicated lncRNAs in the TCGA analyses. The nonparametric Mann–Whitney test was applied due to the non-Gaussian expression distribution and p-value was assigned. Lower part of panel displays the experimental validation of these lncRNAs measured by qRT-PCR and calculated with 2−ΔΔCt method using RPL19 (Ribosomal Protein L19) as endogenous control. Experiments with three biological replicates were performed using two technical replicates for each sample. These results are representative of at least two independent experiments. Values are plotted as expression mean ± Standard Error of Mean (SEM). Unpaired two-tailed t-Test assigned the p-value.