RNA isolation and reverse transcription Shock frozen normal foetal and adult kidneys, ESRD/ACRD kidneys and tumours of the general populations were homogenized in TRIzol (Invitrogen, Germany) as described previously [5]. RNA was isolated according to the manufacturer’s instructions. The concentration of total RNA was measured by spectrophotometry at 260 nm. The quality of RNA was checked by electrophoresis on 1% agarose gel. Reverse transcription of 2 µg RNA was carried out in 25 µl reaction volume with 200 U Superscript II reverse transcriptase (Invitrogen, Germany), 4 µM oligo dT primer, 0.8 mM dNTP, and 40 U RNase inhibitor (Invitrogen, Germany). The reaction was carried out for 60 min at 42°C, followed by 30 min at 50°C.
PCR and quantitative PCR The reaction was performed with 6µl of 1:16 diluted cDNA, 0.133 µM of each forward and reverse primers, 0.2 mM dNTP, 1.5 mM MgCl2, and 0.75 U Taq polymerase (Invitrogen, Germany) over 32 cycles; with denaturation for 30 sec at 94°C, annealing for 30 sec at 61°C, elongation for 45 sec at 72°C and an additional 5 min elongation at 72°C. The PCR product was separated by electrophoresis on 1.5% agarose gel and stained with ethidium bromide. Quantitative PCR was performed using a Real Time PCR Machine (Opticon, MJ Research Inc.). 6 µl of 1:16 diluted cDNA was amplified with 0.133 µM of each forward and reverse primer and 7.5 µl of the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, Germany). Cycling conditions were: 2 min at 50°C and 3 min at 95°C followed by 40 cycles of denaturation for 30 sec at 95°C, annealing for 30 sec 61°C and elongation for 45 sec at 72°C, and an additional 5 min elongation at 72°C. Samples were parallel amplified with gene specific and ß-actin primers. Standard curves were generated from a normal and ESKD kidney cDNA dilution series for both gene specific and ß-actin reactions. The concentration of target genes was calculated by comparing the cycle numbers of the log-linear phase of the samples with cycle numbers of the standards. Data were expressed as the ratio between the amounts of each transcript of interest versus amount of the ß-actin. Melting curves were analysed to determine the specificity of PCRs. Forward and reverse primers used for PCR and real-time PCR analysis were: GPR87 (5’-GGG TTC AAC TTG ACG CTT GCA AAA T-3’ and 5’-GGG TTC AAC TTG ACG CTT GCA AAA T-3’). ß-actin (5’-ATG GAT GAT GAT ATC GCC GCG-3’ and 5’-GTC CAT CAC GAT GCC AGT GGT AC-3’),
Immunohistochemistry Twelve representative slides were selected from six ESRD and six ACRD kidneys removed due to cancer and analysed by immunohistochemistry. The diagnosis of evRCC was established as proposed by Tyckoo et al. [3]. After removing the paraffin and rehydration the 4 µm sections were subjected to heat-induced epitope retrieval in citrate buffer, pH 6.0 in 2100-Retriever (Pick-Cell Laboratories, Amsterdam, The Netherlands). Endogenous peroxidase activity was blocked for 10 min at room temperature. Endogenous peroxidase activity was blocked by incubating the slides with Envision FLEX Peroxydase Blocking Reagent (DAKO) for 10 min. The slides were then incubated for one hour with polyclonal rabbit anti-GPR87 antibody (ab13945, abcam, Cambridge, UK) at the dilution of 1:100 at room temperature. EnVision FLEX horse-radish-peroxidase conjugated secondary antibody (DAKO) was applied for 30 min at room temperature and colour was developed with 3-amino-9 ethylcarbazole (AEC) substrate (DAKO). Tissue sections were counterstained with Mayer's haematoxylin (Lillie’s modification, DAKO) and after 10 seconds bluing mounted with Glycergel (DAKO). Photographs were taken by a Leitz DMRBE microscope, equipped with HC PLAN APO 20x0.70 microscope objective, and a ProgRes C14 camera.