Exp Mol Med.  2013 Sep;45(9):e42.

U6 is unsuitable for normalization of serum miRNA levels in patients with sepsis or liver fibrosis

• Benz F
• Roderburg
• Vargas Cardenas D
• Vucur M
• Gautheron
• Koch A
• Zimmermann H
• Janssen J
• Nieuwenhuijsen L
• Luedde M
• Frey N
• Tacke F
• Trautwein
• Luedde T

Abstract

MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in 'circulating miRNA'. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.

Keyword

miRNA; normalization; serum; spiked-in miRNA; U6

MeSH Terms

Adolescent
Adult
Aged
Aged, 80 and over
Animals
Antigens, Polyomavirus Transforming/blood
Case-Control Studies
Down-Regulation
Female
Humans
Liver Cirrhosis/*blood/diagnosis
Male
Mice
Mice, Inbred C57BL
Middle Aged
RNA, Small Nuclear/*blood
Reference Values
Sepsis/*blood/diagnosis
Antigens, Polyomavirus Transforming
RNA, Small Nuclear