Urinary extracellular vesicles for RNA extraction: optimization of a protocol devoid of prokaryote contamination

Abstract

Background: Urinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established.

Methods: UEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis.

Results: The presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method.

Conclusion: Our results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method must be selected in conjunction with appropriate UEV collection procedure. We also suggested that a minimum 250 ml of urine should be processed to gather enough RNA for robust quantification, qualification and downstream analysis.

Keywords: RNA isolation; RNA quality; dialysis; extracellular vesicles; filtration; microRNA; urine.

Figures

Fig. 1

Workflow of urine collection. (a) Collection of void urine including individual approach for bacterial assessment and pooled for comprehensive analysis of extracellular vesicle RNA. (b) Consecutive collection from individual donors for bacterial presence assessment. 1) Protocol to obtain HFDa; 2) protocol to obtain HFDa0.22 µm; 3) protocol to obtain HFDatablet; 4) protocol of microbiological analysis with HFDa fractions; and 5) differential centrifugations protocol for microbiological analysis.

Fig. 2

Profiles of isolated RNA analyzed by Agilent 2100 Bioanalyzer in PicoChip (electropherograms). (a) UEVs RNA enriched via HFD and isolated with Norgen Kit; (b) cellular RNA extracted with FastRNA Kit; (c) UEVs with spike-in of cellular RNA; (d) cellular RNA re-extracted with Norgen Kit; (e) UEVs RNA, cellular RNA and UEVs+cellular RNA electropherograms merged together according to nucleotide size axis; (f) bacterial RNA; (g) cellular RNA and bacterial RNA mixed and run together; (h) cellular RNA and bacterial RNA electropherograms merged together according to nucleotide size axis. Grey arrow – marker dye; blue arrow – UEVs rRNA; red arrow – 18s and 28s rRNA; black arrow – 16s and 23s rRNA.

Fig. 3

Performance of 0.22 µm filtration and its impact on HFDa fraction (referred as HFDa0.22 µm). (a) Microbiological testing of fractions HFDa0.22 µm experiment performed in triplicate; (b) SDS-PAGE stained with colloidal Coomassie; (c) western blots with anti-ALIX, anti-TSGl0l; and (d) anti-DPPlV, anti-CD63; (e) PicoChip, (f) Small Chip electropherograms from HFDa0.22 µm RNA extracted with Norgen Kit; ST-molecular weight marker, 1,2,3 – HFDa0.22 µm fractions (triplicate), 4,5,6 – SDS elution from Steritop filters (triplicate), kDa – kilodalton.

Fig. 4

Performance of HFD with purification tablet treated SN2,000g (HFDatablet). (a) Microbiological testing of HFDatablet fraction; (b) SDS-PAGE stained with colloidal Coomassie; (c) western blots with anti-ALIX, anti-TSG101; and (d) anti-DPPIV, anti-CD63; (e) transmission electron microscopy picture of vesicles in HFDatablet fraction; lectin blots with (f) MAL II and (g) SNA; (h) Small RNA Chip profiles of HFDatablet and (i) HFDa0.22 µm. ST-molecular weight marker, 1 – HFDatablet, 2 – HFDa0.22 µm, kDa – kilodalton; white asterisk – uromodulin (Tamm-Horsfall protein); red rectangle – MAL II recognized glycoproteins; green rectangle – SNA recognized glycoproteins.

Fig. 5

Pico 6000 RNA Chip electropherograms run in Agilent 2100 Bioanalyzer for RNA samples coming from UEVs extracted with different methods: (a) FastRNA; (b) Qiagen; (c) TRIzol; (d) Norgen; (e) Nucleo-Spin; (f) Quick RNA; (g) mirVana; nt – nucleotide size, grey arrow – marker peak, FU – fluorescence units.

Fig. 6

Small RNA Chip electropherograms run in Agilent 2100 Bioanalyzer for RNA samples coming from UEVs extracted with different methods: (a) FastRNA; (b) Qiagen; (c) TRIzol; (d) Norgen; (e) Nucleo-Spin; (f) Quick RNA; (g) mirVana; nt – nucleotide size, grey arrow – marker peak, FU – fluorescence units.