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.
Highlights
IntroductionMicroRNAs (miRNAs) represent a new class of RNAs that do not transcribe for proteins, but act as regulators of gene expression in multicellular and some unicellular eukaryotes.[1,2] Recently, the involvement of miRNAs was demonstrated in highly regulated processes such as inflammation, fibrosis and carcinogenesis.[3,4,5] Next to their role in the regulation of gene expression in various pathological and physiological processes, serum levels of some miRNAs emerged as biomarkers in different diseases such as inflammation/bacterial infection,[6,7,8] fibrosis[9,10,11] and cancer.[12,13]Because of their low chemical complexity, lack of postprocessing modifications, tissue-specific expression profiles and their extraordinary stability to storage and handling, circulating miRNAs represent potential biomarkers as well. the problem of proper detection of miRNAs was solved with the introduction of standard quantitative real-time PCR (qPCR) assays, the problem of proper data normalization is still unsolved and might explain the high variance between different studies analyzing alterations of circulating miRNAs.[11]
U6 levels display high variability in murine serum U6 serum levels are widely used for the normalization of miRNA-based biomarkers in inflammatory diseases.[14,15,16,17,18]
Variability of U6 serum levels were high compared with levels of spiked-in SV40, representing an alternative, widely accepted method for normalization of miRNA in serum samples.[9,11,23,26,27,28,29,30]
Summary
MicroRNAs (miRNAs) represent a new class of RNAs that do not transcribe for proteins, but act as regulators of gene expression in multicellular and some unicellular eukaryotes.[1,2] Recently, the involvement of miRNAs was demonstrated in highly regulated processes such as inflammation, fibrosis and carcinogenesis.[3,4,5] Next to their role in the regulation of gene expression in various pathological and physiological processes, serum levels of some miRNAs emerged as biomarkers in different diseases such as inflammation/bacterial infection,[6,7,8] fibrosis[9,10,11] and cancer.[12,13]Because of their low chemical complexity, lack of postprocessing modifications, tissue-specific expression profiles and their extraordinary stability to storage and handling, circulating miRNAs represent potential biomarkers as well. the problem of proper detection of miRNAs was solved with the introduction of standard quantitative real-time PCR (qPCR) assays, the problem of proper data normalization is still unsolved and might explain the high variance between different studies analyzing alterations of circulating miRNAs.[11]. MicroRNAs (miRNAs) represent a new class of RNAs that do not transcribe for proteins, but act as regulators of gene expression in multicellular and some unicellular eukaryotes.[1,2] Recently, the involvement of miRNAs was demonstrated in highly regulated processes such as inflammation, fibrosis and carcinogenesis.[3,4,5] Next to their role in the regulation of gene expression in various pathological and physiological processes, serum levels of some miRNAs emerged as biomarkers in different diseases such as inflammation/bacterial infection,[6,7,8] fibrosis[9,10,11] and cancer.[12,13] Because of their low chemical complexity, lack of postprocessing modifications, tissue-specific expression profiles and their extraordinary stability to storage and handling, circulating miRNAs represent potential biomarkers as well. The stable expression of these genes was not validated in larger
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
