Fig. 3

Detection of Somatic retrocopies. (a) Schematic figure representing the RET parental gene and the primer sets (1–7) used to amplify the RET retrocopy. The line represents the non conding sequence (introns and UTR) and the bars represent the exons. The agarose gel shows bands at the expected sizes for intronless copy for all primer sets tested, indicating the presence of a retrocopy of RET in genomic DNA isolated from MTC. As expected, the band corresponding to the intron-containing fragment (parental copy) was observed only for primer set 7 (1868pb). The additional band indentified in line 2 of primer set 2 is an inespecific PCR product (Additional file 2 :Table S2). (b) Representative PCR analysis performed in MTC and control samples using primers located at exons 7 and 9 (primer set 8, Additional file 2: Table S2) of RET gene. The upper band corresponds to the expected PCR product (1498 bp) for RET parental gene and the lower band (238 bp) corresponds to the retrocopy (intronless copy). The lower band is seen only in tumor samples (Lines 1–5) and MTC derived cell lines (lines 9 and 10). Lines 1 and 2 were FFPE derived sample and lines 3–5 are fresh frozen tissue drived samples. The lower band was not seen in peripheral blood DNA from patients (line 6), non-MTC cell line (line 11) and MTC cell line obtained from ATCC (third passage). Positive (line 7) and Negative (line 12) controls were used. (c) Sequencing analysis of the retrocopy band (238 bp) confirming exon-exon junction