Abstract
BackgroundThe consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. We proposed a new in silico approach of finding a suitable reference for human, circulating miRNAs and identified a new set of endogenous reference miRNA based on miRNA profiling experiments from Gene Expression Omnibus. We used 3 known normalization algorithms (NormFinder, BestKeeper, GeNorm) to calculate a new normalization score. We searched for a universal set of endogenous miRNAs and validated our findings on 2 new datasets using our approach.ResultsWe discovered and validated a set of 13 miRNAs (miR-222, miR-92a, miR-27a, miR-17, miR-24, miR-320a, miR-25, miR-126, miR-19b, miR-199a-3p, miR-30b, miR-30c, miR-374a) that can be used to create a reliable reference combination of 3 miRNAs. We showed that on average the mean of 3 miRNAs (p = 0.0002) and 2 miRNAs (p = 0.0031) were a better reference than single miRNA. The arithmetic means of 3 miRNAs: miR-24, miR-222 and miR-27a was shown to be the most stable combination of 3 miRNAs in validation sets.ConclusionsNo single miRNA was suitable as a universal reference in serum miRNA qPCR profiling, but it was possible to designate a set of miRNAs, which consistently contributed to most stable combinations.
Highlights
The consensus on how to choose a reference gene for serum or plasma miRNA expression quantitative polymerase chain reaction (qPCR) studies has not been reached and none of the potential candidates have yet been convincingly validated
We proposed a new design of reference gene selection – employing four different methods of measuring expression stability, we created a framework for identification of reference miRNA sets of a variable number of elements– and tested it on all currently available datasets on Gene Expression Omnibus (GEO) platform to find the optimal set of human serum reference miRNA genes
Single miRNA analysis We found out that mean rankings, calculated from all sets, of miR-222, miR-16 and miR-19b were the lowest
Summary
The consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. MiRNAs represent a group of small non-coding RNA molecules consisting of usually 18–26 nucleotides They regulate gene expression in a sequence-specific posttranscriptional manner and their expression is often altered in diseases and pathological conditions [7, 8]. A major breakthrough in the field of miRNA studies was the observation that they are stably expressed in human serum, and plasma and as such are good candidates for biomarkers of pathological conditions [9, 10]. Such studies typically use a high-throughput method to screen
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