Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.
Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs.
Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993.
Karol L
KL
Thompson
Sanofi R&D, Disposition Safety and Animal Research, Vitry-Sur-Seine, France.
Eric
E
Boitier
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arizona 72079.
Tao
T
Chen
Novartis Pharma AG, Basel, CH 4057, Switzerland.
Philippe
P
Couttet
Toxicology, Bayer Pharma, Wuppertal, AG 42096, Germany.
Heidrun
H
Ellinger-Ziegelbauer
Novartis Pharma AG, Basel, CH 4057, Switzerland.
Manuela
M
Goetschy
Novartis Pharma AG, Basel, CH 4057, Switzerland.
Gregory
G
Guillemain
Astellas Pharma Inc, Osaka 532-8514, Japan.
Masayuki
M
Kanki
AstraZeneca Ltd, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.
Janet
J
Kelsall
Sanofi R&D, Disposition Safety and Animal Research, Vitry-Sur-Seine, France.
Claire
C
Mariet
Institut De Recherches Servier, 78290 Croissy Sur Seine, France.
Catherine
C
de La Moureyre-Spire
Exiqon, Vedbaek DK-2950, Denmark.
Peter
P
Mouritzen
Pfizer, Andover, Massachusetts 01810.
Rounak
R
Nassirpour
ILSI Health and Environmental Sciences Institute, Washington, DC 20005 Rolone@hesiglobal.org.
Raegan
R
O'Lone
National Institute of Standards and Technology, Stanford, California 94305.
P Scott
PS
Pine
Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993.
Barry A
BA
Rosenzweig
AbbVie, Abbott Park, Illinois 60064.
Tatiana
T
Sharapova
Eli Lilly, Indianapolis, Indiana 46285.
Aaron
A
Smith
Takeda Pharmaceutical Co Ltd, Fujisawa, Kanagawa 251-8555, Japan.
Hidefumi
H
Uchiyama
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arizona 72079.
Jian
J
Yan
NIH/NIDDK, Bethesda, Maryland 20892.
Peter S
PS
Yuen
SAS Institute Inc, Cary, North Carolina 27513.
Russ
R
Wolfinger
D000818:Animals; D015415:Biomarkers / Q000097:blood / Q000652:urine; D006335:Heart Injuries / Q000139:chemically induced / Q000378:metabolism*; D007545:Isoproterenol / Q000633:toxicity; D008297:Male; D035683:MicroRNAs / Q000097:blood* / Q000652:urine*; D010949:Plasma / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D044967:Serum / Q000737:chemistry
D015415:Biomarkers; D035683:MicroRNAs; D007545:Isoproterenol
biomarker; interlaboratory; microRNA; variance