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

Then, the expression levels of these genes in serum were analyzed by quantitative reverse transcription PCR (RT-qPCR) among four groups, including patients diagnosed with HCC, chronic hepatitis B (CHB), liver cirrhosis, and healthy subjects. Furthermore, the stability of target genes was evaluated using geNorm, NormFinder, comparative ΔCq programs, and validated by database. We also explored the availability of the miRNA combination, and compared the performance difference between combination and individuals, as well as the selectivity of miRNA references in the combinations. 11 candidate miRNAs were obtained, and miR-4644 stood out among these miRNAs, and proved to be much more stable than other endogenous miRNAs.

Further study showed that miR-4644 exhibited higher stability and expression abundance than other commonly miRNA reference controls. Finally, we discovered the combination of miR-4644 and miR-16 revealed high performance in stability when compared to miRNA individuals. Furthermore, the combination consisted of references with closer nature could give rise to amplification effects in stability.

Our findings demonstrated that miR-4644 is an ideal endogenous normalizer for circulating microRNA quantification in hepatocellular carcinoma. Besides, combining miR-4644 with miR-16 into a whole as a reference control would greatly improve the accuracy of quantification.