Detection of Amplifiable mRNA Extracellular to Insulin-Producing Cells: Potential for Predicting Beta Cell Mass and Function Sweta Rani, Martin Clynes, and Lorraine O’Driscoll * Background: Detecting extracellular nucleic acids in the serum/plasma of cancer patients may help in cancer diag- nosis. We investigated whether extracellular mRNAs are reproducibly detectable in conditioned medium (CM) from insulin-producing cell cultures and if their presence and amounts are indicative of cell number and/or function. Methods: We isolated mRNA from medium condi- tioned by the culture of several insulin-producing cell types: MIN6(L) (glucose-responsive), MIN6(H) (glucose- nonresponsive), and MIN6 B1 murine beta cells and monkey kidney fibroblast cells engineered to produce human preproinsulin (PPI) (Vero-PPI). We used reverse transcription–PCR analyses to evaluate the occurrence of several mRNAs and investigated whether the pres- ence and amounts of the various extracellular mRNAs are associated with cell mass and/or function. Results: Reproducible amplification of mRNAs en- coded by Pdx1, Npy, Egr1, Pld1, Chgb, Ins1, Ins2, and Actb from MIN6(L), MIN6(H), and MIN6 B1 cells and their CM suggests that beta cells transcribe and release these mRNAs into their culture environment. Similarly, PPI mRNA was detected in samples of Vero-PPI cells and CM. The amounts of some mRNAs reflected the numbers and functional status (i.e., glucose responsive- ness vs nonresponsiveness) of the cells conditioning the medium. Although Pax4 mRNA was detected in the MIN6 B1 cell line, the fact that this transcript was not amplifiable from the corresponding CM suggested that mRNA release was selective. Conclusion: mRNAs may be secreted from insulin- producing cells, are reproducibly detected in the extra- cellular environment, and may have potential as extra- cellular biomarkers for assessing beta cell mass and function. © 2007 American Association for Clinical Chemistry Diabetes currently affects 246 million people worldwide and will affect 380 million by 2025 (1). Type 1 diabetes is characterized by autoimmune destruction of the insulin- producing beta cells in the islet of Langerhans of the pancreas, whereas type 2 diabetes is associated with reduced beta cell mass and function, as well as with insulin resistance. No panel of circulating biomarkers of beta cell mass and/or function has been reported. Because type 1 diabetes is usually diagnosed after a loss of 70%– 80% of the insulin-secreting beta cells in the pan- creas (2), detecting an association of gene expression in the circulating serum with beta cell loss and/or function may aid in early disease detection. Nucleic acids were first detected in plasma almost 60 years ago (3), and with the recent advances in sensitive and specific analytical techniques, the detection of nucleic acids now has potential as a tool for early diagnosis of disease. Extracellular RNA has been detected in the plasma and serum of patients with various forms of cancer (4–6), DNA and mRNA of fetal origin have been discovered in the plasma of pregnant women (7–9 ), and significantly increased concentrations of rhodopsin mRNA have been found in the peripheral blood of individuals with diabetes (10 ). Although RNA biomark- ers indicative of pancreas beta cell mass and function may circulate in diabetic patients, no studies have yet investi- gated this possibility. We previously reported methods for detecting mRNAs extracellular to cancer cells (11 ). In an extension of this approach, we investigated whether extracellular mRNAs are reproducibly detectable in beta National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland. * Address correspondence to this author at: National Institute for Cellular Biotechnology, Dublin City University, Dublin 9, Ireland. Fax 353-1-7005484; e-mail Lorraine.ODriscoll@dcu.ie. Received February 21, 2007; accepted August 7, 2007. Previously published online at DOI: 10.1373/clinchem.2007.087973 Clinical Chemistry 53:11 1936 –1944 (2007) Endocrinology and Metabolism 1936