Title: The dynamic microenvironment of pancreatic islet cell transplants precludes the use of common housekeeping genes

Authors: Galit Shahaf; Ali Kassem; Justin B. Levinson; Jared M. Brazg; Eli C. Lewis

Addresses: Faculty of Health Sciences, Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel ' Faculty of Health Sciences, Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel ' Faculty of Health Sciences, Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel ' Faculty of Health Sciences, Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel ' Faculty of Health Sciences, Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, 1 Rager St., P.O. Box 151, Beer Sheva 84105, Israel

Abstract: Background: Pancreatic islet graft survival is affected by early (<3 days) and late immune responses, displaying local CD3+ T cell predominance and loss of insulin production within a week. However, qRT-PCR gene expression profiles require normalisation to housekeeping genes that might be affected by metabolic, inflammatory, hypoxic, apoptotic, revascularisation-related and immune-cell infiltrate changes. Here, we examined eight housekeeping genes. Methods: Syngeneic and allogeneic mouse islet transplantations were performed; day 1 and 7 grafts were analysed for insulin and CD3 transcript levels, as normalised to each housekeeping gene. Results: Normalised qRT-PCR data was not uniform across transplant combinations and time from transplantation. 18s RNA was the superior housekeeping gene, as confirmed by valid changes in CD3 and insulin expression levels. Conclusions: While β-actin and GAPDH are the least-fitting housekeeping genes for the dynamic microenvironment of islet transplants, 18s RNA maintains authenticity across transplant combinations, time from transplantation and altered immune microenvironment.

Keywords: quantitative RT-PCR; islet transplantation; reference genes; syngeneic transplantation; allogeneic transplantation; dynamic microenvironment; pancreatic islet cell transplants; housekeeping genes; pancreatic islet graft survival; immune responses; T cell predominance; insulin production.

DOI: 10.1504/IJIS.2014.066850

International Journal of Immunological Studies, 2014 Vol.2 No.1, pp.42 - 54

Received: 21 Mar 2014
Accepted: 18 Sep 2014
Published online: 10 Jan 2015 *

Full-text access for editors Full-text access for subscribers Purchase this article Comment on this article