A systemic approach to screening high-throughput RT-qPCR data for a suitable set of reference circulating miRNAs

Abstract

Background 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. 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. Results We 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. Conclusions No 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.

NormiRazor: tool applying GPU-accelerated computing for determination of internal references in microRNA transcription studies

“…Stability, in this context, is understood as the invariability of expression of genes or microRNAs between experiments that are subject to changing technical aspects and whose samples may represent different pathological conditions or clinical factors. Although this approach is commonly used (if any systematic procedure of finding reference genes is applied at all), it has recently been questioned by members of our research team [24] as an inadequate method of selecting normalization factors.…”

NormiRazor – Tool Applying GPU-accelerated Computing for Determination of Internal References in MicroRNA Transcription Studies

“…In the present study we proposed a new GPU-based computational approach to the recently published (Pagacz et al, 2020) extensive algorithm searching for reference miRNAs. Since the algorithm is based on an analysis of averaged expression of 2 or 3 miRNA as potential reference, it is highly computationally demanding.…”

“…Another important aspect to consider is the quality of identified references. Previous work of our team showed that averaged expression of 2 or 3 miRNA is a more stable reference than any single miRNA with the measure of stability defined as a score from BK, GN and NF (Pagacz et al, 2020).…”

“…Implementation for combination-based references Following (Pagacz et al, 2020), we redesigned the three most popular normalization algorithms in the transcriptomic community, geNorm, BestKeeper and NormFinder, to facilitate calculations of rankings for combination-based references. At the same time, we reimplemented them in Compute Unified Device Architecture (CUDA) model and encapsulated in a self-contained web application called NormiRazor.…”

Identification of miR-4644 as a suitable endogenous normalizer for circulating miRNA quantification in hepatocellular carcinoma

“…It has been proved to be relatively stable and difficult to degrade in the body fluids, thus, making it the proper reference control for quantification of circulating miRNAs 22 , 23 . Recently, there are numerous reports on the use of endogenous miRNA references for analyzing circulating miRNAs, such as miR-16 24 , miR-24 25 . However, there is still no general consensus until now on reference controls for the normalization of miRNAs quantification in cancers 22 , 26 , let alone for analyzing miRNAs in the serum of HCC.…”