Identification of RNU44 as an Endogenous Reference Gene for Normalizing Cell-Free RNA in Tuberculosis

Abstract Background Normalization of cell-free RNA (cf-RNA) concentration can be affected by variable experimental conditions and thus impact the performance of their diagnostic potential. Our study aimed to identify appropriate endogenous reference genes for cf-RNA biomarker evaluation in the diagnosis of tuberculosis (TB). Methods Subjects consisting of patients with TB with and without malignancy, patients with pneumonia, and healthy controls were recruited. Candidate reference genes were screened and identified by literature reviewing and RNA-Seq analysis. Expression levels of the candidate genes were determined by reverse-transcription real-time quantitative polymerase chain reaction in plasma from patients with TB and healthy controls. The stability of gene expression was assessed by geNorm, NormFinder, BestKeeper, the Comparative Delta Ct method, and RefFinder. Differential expression of 2 small RNAs (sRNAs) encoding by genome of Mycobacterium tuberculosis in plasma of patients with TB were determined by both absolute quantification and relative quantification with candidate reference genes. Results According to the stability ranking analyzed with the 5 computational programs, the top 4 candidates—miR-93, RNU44, miR-16, and glyceraldehyde 3-phosphate dehydrogenase—were used to normalize the transcript levels of 2 mycobacterial sRNAs, MTS2823 and MTS1338, which were observed to have higher copy numbers in the plasma of patients with TB. Normalization with RNU44 displayed significantly higher levels of the 2 M tuberculosis sRNAs in the patients’ plasma than those of healthy controls. Conclusions RNU44 was demonstrated as a proper endogenous gene for cf-RNA normalization in TB diagnosis. RNU44 was demonstrated as a proper endogenous gene for cell-free RNA normalization in tuberculosis diagnosis through RNA-Seq analysis, real-time polymerase chain reaction detection, and computational program evaluation.