The stability among the clades of the phylogenetic tree was assessed by taking 1000 replicates of the dataset and performing analyses using the following programs: SEQBOOT, DNADIST, FITCH, DNAML, DNAPARS, PRODIST, PROTPARS, PROML, and CONSENSE from the PHYLIP software package.
Transmembrane regions of Ce-GnRHR were predicted using the following programs: TMHMM V.2.0 [ 55], DAS-TMfilter [ 56] and PSIPRED [ 57].
Subcellular localization of targeting signals and transmembrane helices of deduced protein-coding genes were predicted using the following programs: PSORT, TargetP, and SOSUI.
Overlapping sequence fragments were concatenated into consensus sequences using Geneious [ 83], and aligned using the following programs (provided as plug-ins in Geneious): MUSCLE for COI [ 84], and MAFFT for 18S and 16S [ 85].
The protein sequences were processed using the following programs: SignalP (version 3.0 and 4.0) (21, 21), Phobius (22), WoLF PSORT (23) and TargetP (24) for secretory signal peptide and subcellular location prediction.
Protein domains and motifs were searched using the following programs and databases: phi- and psi-blast programs of the NCBI platform, SMART [ 32, 33], prosite, pfam, MotifScan, PSORTII, all softwares being available in the Expasy package.
Real-Time PCR was performed in a MyIQ Single-Color Real-Time PCR Detection System (Bio-Rad, Veenendaal, The Netherlands) using the following program: 3 min denaturation at 94°C, 40 cycles of 15 sec at 94°C and 30 sec at 60°C, followed by a melting curve gradient to analyze the specificity of the primer pairs for a particular gene.
PCR was performed for both tumor and normal tissue samples using the following program: 95°C for 15 minutes, followed by 40 cycles of 94°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec.
Reactions for qPCR were prepared using the Corbett robotics machine (Qiagen, UK) and performed on the MJ Research Chromo 4TM (Genetic Research Instrumentation Ltd ,Essex, UK) using the following program: 95°c for 5 minutes, followed by 42 cycles of a 30 second 95°C denaturation, 30 second 61°C annealing and 30 second 72°C extension steps.
PCR was carried out using the following program: 98°C for 30 sec, followed by 15 cycles of 98°C for 10 sec, 60°C for 30 sec and 72°C for 15 sec, and then 72°C for 10 min. The resulting ∼85-bp PCR products were ethanol precipitated and purified from electrophoresis gels using Spin-X filter columns.
Amplifications were carried out using the following program: 95°C for 3 minutes, followed by 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute.
