Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients

Abstract

Chronic kidney disease (CKD) is a severe disorder with an increasing incidence worldwide. An early detection may help to prevent its progression and to minimize the risk of cardiovascular diseases as one of the major comorbidities. Recently, extracellular miRNAs like urinary exosomal miRNAs became of great interest as non-invasive biomarkers which can be determined by RT-qPCR. But until now, there is no consensus regarding the normalization of miRNAs isolated from body fluids. The present study analyzed the miRNAs miR-16, miR-92a, miR-21, miR-124a and the small nuclear RNA RNU6B for their applicability as an endogenous reference gene in expression studies of exosomal miRNAs isolated from CKD patients. For this purpose, miRNA expression levels were determined by RT-qPCR after the isolation of urinary exosomes from 33 CKD patients and from 5 healthy controls. Expression data was analyzed with the normalization determination software NormFinder, BestKeeper, GeNorm and DeltaCt. Our results revealed an abundant expression of the four candidate miRNAs in urinary exosomes and no detectable expression of RNU6B. We identified miR-16 as the most stable endogenous reference gene in our data set, making it a suitable endogenous reference gene for miRNA studies of urinary exosomes derived from CKD patients.

Fig 1. Characterization of urinary exosomes.

Urinary extracellular vesicles were isolated with ExoQuick-TC and prepared for TEM. Vesicles showed an exosome-like shape and size. Scale bar = 100 nm (A). Exosomes of two samples from Norgen urine exosome preparation kit were precipited with ExoQuick-TC and analyzed by Western blot for exosomal markers TSG101, CD9 and CD63. Protein extracts display the presence of exosomal marker proteins over the whole preparation procedure with a slight decrease of band intensities. S = slurry pellet, F = filtrated exosome suspension and R = filtrated exosome suspension after RiboShredder treatment (B). Zetasizer analysis of urinary exosome samples (C). The results of the 5 measurements of the urine sample displayed in TEM (A) are shown (C, a). Beside this the results of the 5 measurements per sample of three urinary exosome samples are shown (C, b). The figures show vesicles with typical exosomal size and some bigger structures that might be due to clumping artefacts.

Fig 2. Expression distribution and stability of normalization candidate mature miRNAs.

All four candidate miRNAs are abundantly expressed in urinary exosomes of CKD patients and the normal group. Exosomal RNA was isolated with the Norgen urinary exosome preparation and RNA isolation kit. Expression data was generated by qRT-PCR using Taqman® miRNA Assays. The raw Ct-values were normalized against RNA input and an inter-run calibrator (A). MiR-92a is the most stable candidate normalizer within the data set, as revealed by NormFinder software. The best combination is miR-92a/16 with miR-16 as the most stable one, when input data is split into CKD patients and normal group. Error bars = SD (B).

Fig 3. Stability of normalization candidate mature miRNAs determined with different algorithms.

Stability values were calculated by the online available tools BestKeeper, DeltaCt, Genorm and NormFinder. The lower the stability value, the higher the stability. Each tool reveals miR-92a as the most stable one or the combination of miR-92a/miR-16. miR-92a = yellow, miR-16 = blue, miR-21 = purple and miR-124a = green.

Fig 4. Expression differences of the normalization candidates between healthy controls and chronic kidney disease (CKD) patients.

Box-Whisker Plot of the RNA input- and inter-run calibrator-normalized expression values determined by Taqman® qRT-PCR. The midline represents the median and the box borders represent the inter-quantile range. Percentiles are displayed by whiskers.