Strand-specific GHRLOS real-time RT-PCR. (A) Strand-specific real-time RT-PCR approach to quantify GHRLOS expression. To select for RNA generated from the antisense strand, a linker sequence (LK) (not present in the genomic sequence of GHRLOS) is attached to a GHRLOS gene specific primer and employed in reverse transcription. The resulting cDNA is then combined in a real-time PCR, combining a gene-specific primer (GSP) and a primer containing the linker region from reverse transcription primer only. An internal Taq Man probe (depicted as a blue box) was employed to increase the specificity and sensitivity of the assay. (B) Relative total GHRLOS expression in a range of human human tissues. (C) Relative total GHRLOS expression in human cell lines (U-937 non-Hodgkin's lymphoma, SW1353 chondrosarcoma, CaCo-2 colorectal adenocarcinoma, SAOS-2 osteosarcoma, U-87 MG and U-251 MG glioblastoma, HEK293 human embryonic kidney and OVCAR-3 ovarian cancer). Calculations of GHRLOS expression levels were performed using the standard curve method (correlating the threshold cycle number (CT values) and copy numbers of GHRLOS), and normalised to the expression of 18S ribosomal RNA. Each bar presents the mean ± standard deviation of duplicate reactions.