Facturers manual, using approximately 50 mg frozen tissue per milliliter TRIzol.Real-time
Facturers manual, using approximately 50 mg frozen tissue per milliliter TRIzol.Real-time quantitative RT-PCR AnalysisProtein concentration was measured in triplicate by the method of Bradford, using BSA (1 mg/mL) as the calibrator. The results were recorded as units PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25112874 of peptidase (UP) per milligram of protein. One unit of peptidase activity is the amount of enzyme necessary to release one pmol of fluorogenic product per minute (pmol/min). Fluorogenic assays were linear with respect to hydrolysis time and protein content.Quantitation of DPP IV and NEP mRNA expression RNA isolationQuantitative RT-PCR for detecting DPP IV and NEP mRNA (DPP4 and MME genes respectively) was performed to assess the transcription levels of these peptidases. To avoid any RNA degradation only samples that were immediately immersed in RNAlater after dissection were used in these experiments. Hence, total RNA of tumor and non-tumor tissue samples from 26 CCRCCFirst-strand cDNA was synthesized from 25 g of total RNA of each human sample using Moloney murine leukemia virus reverse transcriptase and random hexamers according to the manufacturer’s instructions (First-strand cDNA Synthesis Kit, Amersham Biosciences, Essex, UK). The resulting cDNA samples were amplified by PCR with specific oligonucleotide primer pairs designed with the analysis software Primer 3 [20]. Based on previous experiments on human renal cell carcinoma [19,21] and other human tissues [22,23] we chose TATA box binding protein (TBP), peptidylprolyl isomerase A (PPIA), -actin (ACTB) and succinate dehydrogenase complex, subunit A (SDHA) as endogenous reference genes. The sequence of the primers used to amplify DPP4, MME and the four housekeeping genes are shown in Table 1. All primers were synthesized and purified by Sigma-Genosys (Cambridge, UK). The expression of the housekeeping genes, DPP4 and MME was quantified in all cDNAs by real-time PCR using the iCycler iQ real-time detection apparatus (BioRad Laboratories, Hercules, CA, USA). Dilutions of the cDNA template were prepared from each tissue and amplified in triplicate using SensiMix Plus SYBR + FLUORESCEIN (Quantace Ltd., London, UK). Three purchase JWH-133 negative controls (with no template, no PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28667899 reverse transcriptase and no RNA in the reverse transcriptase reaction) were alsoTable 1: Sequence of forward (F) and reverse primers (R) of indicated target genes and the size expected for each PCRamplified product.Peptidase DPPIV/gp110 NEP/CALLA glycoprotein Housekeeping Gene name -actin Succinate dehydrogenase complex, subunit A TATA box binding protein Peptidylpropyl isomerise A ACTE SDHA TBP PPIA 5′-TCCCTGGAGAAGAGCTACGA-3′ 5′-TCTGCCCACACCAGCACT-3′ 5′-GGATAAGAGAGCCACGAACCAC-3′ 5′-GGTCCCAAAGACAGCAGAAAA-3′ 5′-ATCTGCTGGAAGGTGGACAG-3′ 5′-CCTCTCCACGACATCCTTCC-3′ 5′-TTAGCTGGAAAACCCAACTTCTG-3′ 5′-TCACCACCCTGACACATAAACC-3′ 362 142 139 114 Gene symbol Fordward primer DPP4 MME 5′-AGTGGCGTGTTCAAGTGTGG-3′ 5′-CCGAGAAAAGGTGGACAAAGA-3′ Reverse primer 5′-CAAGGTTGTCTTCTGGAGTTGG-3′ 5′-GGACTGCTGGGCACTAAAGAA-3′ Amplicon size (bp) 112Primers for the assayed housekeeping genes are also shown.Varona et al. BMC Cancer 2010, 10:193 http://www.biomedcentral.com/1471-2407/10/Page 4 ofincluded in each plate to detect any possible contamination. After a hot start (10 min at 94 ), the parameters used for PCR amplification were: 10 s at 94 , 20 s at 60 and 30 s at 72 , for 50 cycles. Real-time PCR data were expressed as the fold change of the target gene expression relative to the geometric.
