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Fig. 1 | Clinical Proteomics

Fig. 1

From: Integrative omics reveals subtle molecular perturbations following ischemic conditioning in a porcine kidney transplant model

Fig. 1

Overview of transcriptomic and proteomic workflows. A For transcriptomics, tissue samples were cut, weight and lysed. RNA was extracted, followed by cDNA library preparation, and the difference in transcript level determined between non-RIC and RIC groups. For selected targets of interest, qPCR was undertaken. B In the proteomics arm, proteins were extracted, reduced, alkylated and subjected to methanol/chloroform extraction, digested with trypsin, cleaned-up and subjected to 10plex TMT labelling. Labelled peptides were separated by high pH-RP, prior to analysis by LC–MS/MS. The green boxes outline our sample pooling and fractionation strategy. A total of 16 tissue samples (and 4 pools) were labelled across two TMT kits, with 8 RIC and non-RIC pairs and 2 pools per 10plex kit. Each TMT experiment was subsequently separated into 100 fractions, which were then concatenated down to 20 fractions. This resulted in a final total of 40 samples for injection into the mass spectrometer

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